Perfusion technologies provide a means to assess the quality of an organ prior to transplantation. This can aid in the decision process, preventing the likelihood of primary nonfunction and also the unnecessary discard of the organ. Hypothermic perfusion techniques have proved useful in determining the level of injury but perfusion parameters (flow and resistance) are poor predictors of outcome and cannot reliably discriminate between organs that should or should not be transplanted.1
Normothermic perfusion technologies have significant advantages over hypothermic techniques. Graft function is restored and the exposure to cold ischemia is lessened. Furthermore, an assessment of functional parameters during perfusion not only allows a measure of injury but also provides an evaluation of recovery. We have previously described the application of an ex vivo normothermic kidney perfusion (EVKP) quality assessment score in clinical transplantation.2 The assessment score is based on 3 basic parameters during perfusion: macroscopic appearance, renal blood flow and urine output. Kidneys are scored from 1 (least injury) to 5 (the most severe injury). Nonetheless, other markers of function and injury may also be incorporated into the assessment to provide a more comprehensive measure of quality.
Urinary biomarkers are emerging as an important diagnostic aid in the early detection of acute kidney injury (AKI) in a wide range of settings.3-5 Neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) are 2 of the most recognized and reliable markers of kidney injury and point-of-care devices are available to provide results for both within 10 to 15 minutes. Endothelin-1 is a nonrenal specific marker of tissue injury. Nevertheless, we have previously found it to be a reliable index of tubular injury in a porcine model.6,7
The aim of this study was to examine the relationship between urinary biomarkers (endothelin-1 [ET-1], NGAL and KIM-1) and EVKP parameters in a series of human kidneys that were rejected for transplantation in the UK.
MATERIALS AND METHODS
From December 2012 to January 2014, 56 kidneys from the national organ allocation scheme that were deemed unsuitable for transplantation were recruited in this research project. Consent for the use of the organs for transplantation and research was obtained from the donor family by the Specialist Nurses in Organ Donation before organ retrieval. Ethical approval was granted for the study by the national research ethics commission in the UK, National Research Ethics System (12/EM/0143).
Kidneys were retrieved by the UK National Organ Retrieval Service as previously described.2 The kidney donor information was recorded and included donor age, gender, donor type and terminal serum creatinine levels. The warm and cold ischemic times were also recorded.
Ex Vivo Normothermic Perfusion and Functional Analyses
Kidneys were prepared for EVKP and the renal artery, vein, and ureter cannulated. The EVKP circuit was primed with a perfusate solution (Ringer's solution; Baxter Healthcare) and supplements added to provide a physiological environment.8,9 One unit of group 0 positive packed red blood cells from the local blood bank was added to the priming solution.
Kidneys were perfused with the red cell based solution at a set mean arterial pressure and near normal body temperature. Supplements were infused into the venous reservoir and arterial arm of the circuit to maintain normal homeostatic conditions as previously described.2,8-10
Creatinine was added to the red cell based perfusate to allow an assessment of creatinine clearance and tubular function. The renal blood flow and mean arterial pressure were recorded continuously and the mean calculated. Urine output was collected throughout perfusion and the total output measured. Blood gas analysis of arterial and venous blood was used to record acid-base homeostasis and measure oxygen consumption and extraction. Blood samples were taken preperfusion and after 60 minutes of EVKP. A urine sample was collected after 60 minutes of EVKP. Percentage creatinine fall and fractional excretion of sodium were calculated and recorded.
Urine levels of ET-1 were determined using an enzyme immunometric assay kit (Assay Design, Ann Arbor, MI). Levels of NGAL and KIM-1 were measured using a human NGAL sandwich enzyme-linked immunosorbent assay (ELISA) kit (BioPorto Diagnostics, Gentofte, Denmark) and KIM-1 ELISA kit (Enzo Life Sciences, Exeter, UK). The samples and standards were added in duplicate to the precoated plates and the assays carried out as per the manufacturer's instructions.
EVKP Assessment Parameters
Each kidney was categorized into 3 groups according to its macroscopic appearance during EVKP as follows.2
Grade I: excellent perfusion (global pink appearance—1 point).
Grade II: moderate perfusion (patchy pink/purple appearance which either remained or improved during EVKP—2 points).
Grade III: poor perfusion (global mottling and purple/black appearance which remained throughout EVKP—3 points).
Derivation of EVKP Assessment Score
A combination of the macroscopic and functional parameters was used to create an index of organ quality. Receiver operating characteristic (ROC) curves were previously used to determine thresholds of renal blood flow and urine output to differentiate between macroscopic grades I and II versus grade III.8 These thresholds were combined with the macroscopic grade to give an overall EVKP assessment score of 1 to 5. Macroscopic grades I, II and III were assigned points of 1, 2 and 3, respectively. Kidneys were assessed by 2 assessors blinded to the donor details. Kidneys with a mean renal blood flow below the threshold (<50 ml/min per 100 g) were given an additional score of 1. Kidneys producing less than the threshold of urine output (<43 mL in 60 minutes) were also given an additional score of 1. Therefore, overall EVKP assessment scores ranged from 1 indicating the least injury to 5, the most severe. This score provides a quantitative measure of functional parameters that supports the macroscopic appearance.
A wedge renal biopsy was taken on arrival at the laboratory after the period of static cold storage. The tissue was fixed in 10% formal saline then embedded in paraffin wax. Sections from the paraffin embedded tissue were cut (4 μm) and stained with hematoxylin-eosin for histopathological scoring.
A consultant pathologist graded the sections using the Remuzzi score and assessed the level of acute tubular injury (ATI). Sections were graded mild, mild to moderate, moderate, and severe for the presence of ATI.
Continuous data are presented as mean ± SD. Data were compared using the Kruskal-Wallis test with Dunn multiple comparisons test where appropriate. Categorical variables were analyzed by χ2. A P value less than 0.050 was considered statistically significant. Donor factors (age, retrieval creatinine, the warm ischemic time, and cold ischemic time) and perfusion parameters (The EVKP score, renal blood flow, oxygen consumption, oxygen extraction, urine output, percentage creatinine fall, and fractional excretion of sodium) were correlated with the urinary biomarkers score using linear regression (Pearson). The Kidney Donor Risk Index (KDRI) was calculated11 and correlated with the EVKP score. GraphPad Prism 6 was used for statistical analysis (GraphPad Software, La Jolla, CA).
Fifty-six kidneys from 41 donors were included in the study. The kidneys were declined for transplantation due to the following reasons: donor history (n = 12), poor in situ flush (n = 9), hypothermic machine perfusion parameters (n = 6), anatomical/technical reasons (n = 14), suspected malignancy (n = 6), AKI (n = 1), histology score (n = 3), fat embolism (n = 2), infection (n = 1), prolonged cold ischemia (n = 1), and no suitable recipient (n = 1). The EVKP scores were as follows: 22 scored 1, 16 scored 2, 8 scored 3, 4 scored 4, and 6 scored 5. To simplify analysis, the kidneys were grouped as follows: group A, EVKP score 1 or 2 (n = 38); group B, EVKP score 3 (n = 8); and group C, EVKP score 4 or 5 (n = 10).
There were significantly fewer female donors in the EVKP group B and C kidneys compared with group A (P = 0.034; Table 1). In the EVKP group B kidneys, all the donors were from donation after brain death (DBD) donors, whereas in EVKP group C, all were from donation after circulatory death (DCD) donors (P = 0.001; Table 1). There was no significant difference in the duration of warm or cold ischemia (P = 0.581, 0.051, respectively). The terminal serum creatinine was significantly higher in EVKP group C kidneys compared with group A (P = 0.001; Table 1). Nine of the 10 donors in EVKP group C kidneys had a raised terminal serum level (>121 μmol/L).
Perfusion Parameters and Urinary Biomarkers
Kidneys in EVKP group A had improved perfusion parameters compared to groups B and C. The renal blood flow, oxygen consumption, and urine output were significantly higher in group A compared with groups B and C (P <0.05; Table 2). Serum creatinine fall was also significantly greater in group A compared with group C (P <0.05; Table 2). There was no significant difference in the level of fractional excretion of sodium between the groups although, levels were numerically higher in group B and C kidneys (P = 0.317; Table 2).
Levels of ET-1 were significantly higher in EVKP group B kidneys compared with group A (P = 0.008; Figure 1A). Although, numerically, levels were the highest in EVKP group B kidneys, NGAL was significantly lower in EVKP group A kidneys compared with group C (P = 0.018; Figure 1B). There was no significant difference in the urinary levels of KIM-1 between the groups (P = 0.649; Figure 1C).
Higher urinary levels of ET-1 and NGAL were associated a higher EVKP assessment score (R2 = 0.1771; P = 0.001, R2 = 0.0997; P = 0.018, respectively). There was no correlation with the assessment score and KIM-1 (R2 = 0.00532; P = 0.600).
Lower urinary levels of NGAL were associated with a higher mean renal blood flow (P = 0.008; R2 = 0.1890), higher level of oxygen consumption (P = 0.004; R2 = 0.1440), increased urine output (P = 0.049; R2 = 0.070), and decrement in creatinine (P = 0.0013; R2 = 0.1850). Lower urinary levels of ET-1 were associated with a greater decrement in creatinine (P = 0.001; R2 = 0.1938). There was no significant correlation between the urinary levels of KIM-1 and any of the perfusion parameters.
Correlation of Perfusion Parameters With Donor Demographics
There was no association with the donor age or length of warm or cold ischemia and any of the perfusion parameters (P > 0.05). Kidneys from female donors were associated with higher urine output (P = 0.0001; R2 = 0.2367), a greater decrement in creatinine (P < 0.0001; R2 = 0.2624), and a lower EVKP score (P = 0.001; R2 = 0.1528). A higher serum creatinine level before retrieval was associated a lower level of renal blood flow, oxygen consumption, higher oxygen extraction, and a lower level of renal function during EVKP (P > 0.05; Table 3). It was also associated with a higher EVKP score. There was no association with the level of fractional excretion of sodium and any of the donor demographics (P = 0.4822; Table 3). The KDRI score did not correlate with the EVKP score (R2 = 0.0010; P = 0.820).
Correlation of ET-1, NGAL, and KIM-1 With Donor Demographics
Higher levels of urinary ET-1 and NGAL were associated with a raised creatinine before retrieval in the donor (R2 = 0.1139; P = 0.014 and R2 = 0.1385; P = 0.007, respectively). However, there was no association with donor age, warm ischemic time or the cold ischemic time (P > 0.05). There was no association with KIM-1 and donor demographics or creatinine levels before retrieval (R2 = 0.02458; P = 0.272).
Donation After Circulatory Death Versus Donation After Brain Death Kidneys
The cold ischemic time was significantly longer in the DBD kidneys compared with the DCD (P = 0.026; Table 4). Urinary levels of ET-1 were significantly lower in the DCD kidneys compared with DBD (P = 0.043; Table 4). There were no other significant differences between the donor demographics, perfusion parameters, and urinary biomarkers (Table 4).
Thirty-two biopsies were available from EVKP group A kidneys, all of group B kidneys and from 7 of 10 group C kidneys. The median Remuzzi score was 4 (range, 0-10) in EVKP group A kidneys, 5 in group B (range, 1-10) and 3 (range, 0-6) in group C kidneys. Eleven of 32 of EVKP group A kidneys had mild acute tubular injury (ATI) 5 mild to moderate, and 2 moderate ATI. One kidney in EVKP group B had mild, 1 mild to moderate, and 1 moderate acute tubular injury (ATI). Two kidneys in EVKP group C kidneys had mild ATI, and 2 had moderate ATI. There was no association with urinary levels of NGAL, KIM-1, ET-1, or any of the perfusion parameters, the Remuzzi score or the level of AKI (P > 0.05).
This study demonstrates that levels of urinary ET-1 and NGAL measured after 60 minutes of EVKP were significantly correlated with perfusion parameters during EVKP. These biomarkers and EVKP perfusion parameters were also significantly correlated with terminal renal function in the donor. There was no association between urinary KIM-1 after EVKP and either perfusion parameters or terminal renal function in the donor. The measurement of biomarkers during EVKP provides additional information about donor organ quality.
Normothermic perfusion techniques are evolving as an important asset in the decision process to determine the suitability of an organ for transplantation. We have previously reported that basic functional parameters (renal blood flow and urine output) together with an assessment of the macroscopic appearance can be used to formulate a kidney quality score.2 Evidence so far suggests that a higher EVKP score is associated with an increased risk of delayed graft function (DGF).2 This technology has also been used to assess human kidneys declined for transplantation due to poor in situ perfusion at the time of retrieval.10 We have subsequently gone on successfully transplant 2 of these kidneys based on this assessment.12 This present study has shown that other measures of function including the rate of oxygen consumption, oxygen extraction, and creatinine fall can be used to provide a more detailed assessment of function and quality.
Measurements of serum creatinine are used clinically to assess the level of renal function. Nonetheless, creatinine levels are affected by other physiological functions and are regarded as a late marker of renal injury.4 Urinary biomarkers, particularly NGAL and KIM-1, are sensitive measures of kidney injury and can be used to provide a more accurate assessment.4-6,13,14 In the posttransplant setting, they have a high predictive value and are strongly associated with graft dysfunction. More recently Koo et al15 measured several biomarkers (NGAL, KIM-1, and liver fatty acid binding protein [L-FABP]) in a series of 94 deceased donors to determine kidney quality and outcome. They used a multiple logistic regression model to generate a prediction score. Independently, NGAL and L-FABP were associated with AKI in the donor and predictive of reduced graft function in the recipient [area under the curve ROCs, 0.758 and 0.704, respectively]. When included in a scoring system their predictive value was greater (area under the curve ROC, 0.808). In a larger series of 1304 deceased donors, Reese et al16 also found that higher levels of urinary NGAL were strongly associated with donor AKI and had some association with delayed graft function (DGF) in the recipient. However, their ability to predict primary nonfunction and determine which organs should be transplanted or discarded was low. Hollmen et al17 showed that in donors with a urinary NGAL of 18 ng/mL or greater, DGT was more prolonged and graft survival reduced. Interestingly, in all these studies, KIM-1 had no association with donor AKI or outcome. NGAL is an earlier marker of kidney injury upregulated within hours, whereas KIM-1 is reported as a later marker, although the literature is conflicting.18 van den Akker et al19 found that KIM-1 was not detectable in the perfusate of the graft after preservation or until day 4 posttransplant. In contrast, Field et al recently reported that KIM-1 was a promising marker of acute kidney injury in a series of deceased donors.20 Biomarkers have also been used during hypothermic machine perfusion in combination with flow and resistance parameters to determine quality. However, individually, they had low a predictive value and could not distinguish between transplanted and discarded kidneys.21
The third biomarker used in this study, ET-1, is involved in regulating renal vascular tone and tubular secretion of electrolytes and water. Clinically, high urinary levels have been associated with chronic kidney22,23 and polycystic kidney disease.24 In a porcine model of reperfusion injury, we previously found that elevated levels of ET-1 accurately reflected the level of ischemic injury.6,7 More recently, ET-1 has been used as a marker of lung injury during ex vivo lung perfusion (EVLP).25 Perfusate levels predicted lung function and were associated with outcome after transplantation.
Levels of fractional excretion of sodium, a marker of tubular function, and urinary KIM-1 were not significantly different across the grades of kidneys and not associated with donor AKI. Therefore, their use in this setting appears to be limited. The histology revealed a wide variation in the level of donor related damaged. A significant proportion of kidneys had evidence of AKI. Renal function is not necessarily associated with histological findings,26 and we found no significant correlation between the Remuzzi score, urinary biomarkers, donor demographics, or EVKP parameters. This is in agreement with Koo et al15 who found no association with the biopsy injury score, NGAL, or outcome in their series of deceased donor kidneys. The Remuzzi score is a histological assessment of chronic renal damage, whereas EVKP provides functional information about ATI. The lack of correlation between the 2 is therefore unsurprising. This argument applies equally to the KDRI.
The role of urinary biomarkers has not been previously investigated during EVKP of human kidneys. Although there is no follow-up data on outcome, this study provides important information for the future development of EVKP as an assessment device. Our described technique of EVKP is carried out at the end of the preservation interval for 60 minutes before transplantation. The technique requires some added resources but is straightforward and brief. We found that kidneys with a higher EVKP score, indicating lower quality also had reduced functional parameters and higher levels of urinary NGAL and ET-1. These parameters were also associated with raised levels of serum creatinine recorded in the donor before organ retrieval. Although this suggests that these biomarkers are reliable indices of ATI which may aid in the assessment of a kidney, it also raises the question of the cost effectiveness and extra utility of measuring biomarkers in addition to the currently available donor data and EVKP assessment. The biomarkers are typically measured using ELISA techniques, although, a point-of-care devise is available for NGAL which would makes its application into clinical practice relatively straightforward.
Larger series are required to determine whether these biomarkers can be used as a reliable assessment tool in combination with EVKP to predict graft outcome. We are currently performing a multicenter randomized controlled trial comparing static cold storage with EVKP in DCD kidney transplantation which is due to be completed in 2020 (ISRCTN15821205). Urine samples are being collected during EVKP (n = 200) for the analysis of a range of biomarkers including NGAL, ET-1, and L-FABP. These will be correlated with transplant graft outcome, donor demographics, EVKP parameters, and histological analysis. This is likely to provide us with a more conclusive evaluation of their use in this setting.
A limitation to the described EVKP technique is perhaps the short duration. Of note during the 60 minutes of perfusion, there was no association between any of the perfusion parameters or urinary biomarkers and the level of ischemic injury. This may be explained by the duration and conditions necessary for these effects to become evident. Warm and cold ischemic injuries have a detrimental effect on graft function and outcome. The reintroduction of oxygenated blood mediates a series of injury processes involving activated endothelial cells causing vasoconstriction, the recruitment of leukocytes to the injured tissue leading to failure of the microcirculation and the “no-reflow” phenomena.27 There is also a release of damage associated molecular pattern molecules which activate reactive oxygen species, molecular, and signalling cascades.28 These events cause significant tissue injury which results in AKI and DGF. Although circulation is restored during EVKP, the conditions are designed to be protective. The absence of white cells, platelets and complement factors abrogate graft injury, and therefore, it is perhaps unsurprising that the impact of ischemic injury appears to be undetected during this time. Nonetheless, longer perfusion times may provide additional information on the impact of ischemic injury.
In conclusion, EVKP continues to be developed as a comprehensive pretransplant assessment technology/tool. The measurement of urinary biomarkers during EVKP undoubtedly provides additional information but it is not clear at this point whether or not this will improve our ability to select marginal kidneys for transplantation. Because this was a preliminary study of a relatively small number of discarded human kidneys, further analysis from the randomized controlled trial will evaluate the potential value of biomarkers in predicting clinical allograft outcomes.
The authors would like to thank Miss Meeta Patel for her technical help and Dr. John Dormer for performing the histology analysis.
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