Orthotopic liver transplantation (OLT) is unavoidably associated with ischemic injury, a condition leading to acute cellular injury, subsequent activation of immunological and inflammatory processes, and organ injury.1 Furthermore, ischemic injury induces pathophysiological alterations in the endothelium, most prominently degradation of the endothelial glycocalyx.1-3 Syndecan-1 (Sdc-1), a transmembrane heparan sulfate proteoglycan, is the main component of the endothelial glycocalyx and widely used as a glycocalyx biomarker, reflecting the extent of endothelial glycocalyx destruction.4 Elevated Sdc-1 concentrations have been observed within lung and kidney grafts during ischemic organ preservation.5,6 Although the detrimental effect of ischemic organ preservation on liver grafts has been documented in the clinical setting of OLT,7,8 Sdc-1 has never been assessed in the effluent of human liver grafts.
To overcome the actual problem of donor organ shortage, liver grafts are increasingly being collected from extended criteria donors. However, livers derived from extended criteria donors are frequently considered as grafts with potentially lower graft quality. Transplantation of liver grafts with lower quality severely impacts postoperative graft function.9-11 Impaired graft function occurring within 1 week after OLT is clinically summarized as early allograft dysfunction (EAD), a condition affecting ~20% of transplanted patients.9,12,13 Development of EAD negatively impacts outcome, eventually leading to urgent retransplantation or even death.9,12,14 EAD is diagnosed after OLT based on highly elevated serum transaminases, persistent coagulopathy, and cholestasis.15 Prediction of EAD at earlier time points could improve outcome after OLT. Biomarkers within the liver graft effluent have been shown to predict postoperative graft dysfunction and graft survival.16-18 Thus, analysis of biomarkers within effluent might allow for direct assessment of graft quality before transplantation.
The primary aim of this study was to assess the extent of glycocalyx degradation within the human liver graft. In addition, effluent Sdc-1 concentrations were correlated with effluent concentrations of hepatic injury markers to evaluate whether glycocalyx degradation is associated with liver graft injury. The secondary aim was to associate Sdc-1 concentrations with EAD, a substitute for graft quality. In addition, we assessed whether effluent and serum Sdc-1 concentrations are associated with 1-year graft survival and 1-year patient survival after OLT.
MATERIALS AND METHODS
Samples collected during our prospective, observational pilot study registered at clinicaltrails.gov (NCT02695979)1 were analyzed for this investigation. The study was approved by the institutional ethics committee (MUV 2171/2014 and 2026/2015) and performed in accordance with the ethical standards laid down in the declaration of Helsinki at the General Hospital of Vienna. All patients signed written informed consent before participation. Thirty-eight patients who underwent OLT due to end-stage liver disease (ESLD) between August 2014 and August 2015 were included. Exclusion criteria were defined as the need of veno-venous bypass during OLT, combined liver-kidney transplantation, and high urgency transplantation.
Graft Preservation and Surgery
All grafts were ABO identical and matched for the physiological body-to-weight ratio of recipients. All liver grafts were retrieved from brain-dead donors and preserved in a histidine-tryptophan-ketoglutarate solution (Custodiol, Koehler; Bensheim, Germany). Before reperfusion, liver grafts were flushed with 1000 mL sterile 5% albumin solution to remove the preservation solution and other molecules that accumulated during hypoxic storage.
Surgery was performed using the total caval replacement technique without a veno-venous bypass. Reperfusion was started via the portal vein. Biliary anastomosis was performed as a duct-to-duct anastomosis. A biliary drainage was not routinely placed, and intra-abdominal drainages were only placed in high-risk patients. Surgical techniques including partial clamping of the caval vein combined with temporary porto-caval shunting and piggy-back or side-to-side cavo-cavostomy are not routinely performed at our center. All patients were postoperatively admitted to the intensive care unit, and immunosuppression was administered according to the local standardized protocol for immunosuppression in OLT, as described elsewhere in detail.4,19
Sample Collection and Measurements
Liver graft effluent samples were collected from the inferior caval vein while flushing the organ with 1000 mL 5% albumin solution via the portal vein. The effluent is defined as the washout of the organ preservation solution.20 In addition, serum samples were drawn from a radial arterial catheter at following time points: before surgery at baseline (BL), at the end of OLT at day 0 (D0), and at day 1 (D1), day 2 (D2), and day 3 (D3) after OLT. All samples were spun at 3000 rpm for 15 minutes immediately after collection and stored in cryotubes at −80°C until analysis.
In all collected samples, concentrations of Sdc-1 were determined by enzyme-linked immunoassay, according to the manufacturer’s protocol (Diaclone Research; Besancon, France). In addition, concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), lactate dehydrogenase, creatine kinase, alkaline phosphatase (AP), and lactate19 were measured in the effluent samples to assess the extent of liver graft injury.
Effluent Sdc-1 concentrations were correlated with effluent concentrations of ALT, AST, lactate dehydrogenase, creatine kinase, AP, and lactate to evaluate whether the levels of Sdc-1 released by the liver graft is proportional to the extent of liver graft injury. In addition, effluent Sdc-1 concentrations were correlated to cold and warm ischemia times to assess whether Sdc-1 release by the graft is related to ischemia time.
Classification of EAD
EAD was defined according to a validated clinical definition as the presence of any of the following laboratory criteria9: (1) serum ALT or AST concentrations >2000 IU/L within the first 7 days after transplantation, (2) serum bilirubin concentrations ≥ 10 mg/dL on day 7 after transplantation, and (3) international normalized ratio ≥1.7 on day 7 after transplantation. Patients who met the criteria for EAD within 7 days after OLT were compared with patients with normal postoperative graft function.
Sample size calculation was based on our previous study.19 Statistical analyses were performed with Prism 6.0 (GraphPad Software, La Jolla, CA) and SPSS Statistics 25 (IBM, Armonk, NY). A Kolmogorov-Smirnov test was used to assess normal distribution of data. Normally distributed data are depicted as mean with SD; data not normally distributed are depicted as median with interquartile ranges (IQR [25%–75%]). Correlations were analyzed with Spearman or Pearson analysis. T tests or Mann-Whitney U tests with correction for multiple comparisons were used to compare time points. Differences between various time points in different groups were analyzed using parametric or nonparametric ANOVA with multiple comparisons. To determine whether Sdc-1 is associated with postoperative graft function or survival, receiver operating characteristics (ROC) curve analyses with positive predictive values (PPV) and negative predictive values (NPV) as well as univariate regression analyses with EAD, 1-year graft survival, and 1-year patient survival as outcome factors were performed. In addition, multivariate regression analyses were performed for all 3 outcome measures, including parameters showing significant effects in univariate analysis and parameters of clinical importance as covariates. For all analyses, a P < 0.05 was considered significant; adjusted P values are reported when data have been corrected for multiple comparisons.
Thirty-eight (30 male and 8 female) patients undergoing OLT were included in this study. The mean age of all patients was 57 ± 9 and their preoperative model of end-stage liver disease score was 17 ± 5. Etiology of ESLD was mainly alcoholic cirrhosis (16 patients) and viral hepatitis (9 patients). Hepatocellular carcinoma (4 patients), autoimmune hepatitis (4 patients), primary biliary cirrhosis (1 patient) as well as undefined cirrhosis (4 patients) were further causes leading to ESLD. All pat ients were of Caucasian ethnicity.
Within the first 7 days after OLT, 5 patients developed postoperative complications. These patients were classified as grade IIIb according to the Clavien-Dindo classification21,22 and required surgical revision. All patients survived until discharge from the intensive care unit. The mean hospital length of stay of all patients was 22±12 days. Of the 38 included patients, 7 (18%) met the criteria for EAD within the first week after OLT. Two of the 7 EAD patients suffered postoperative complications requiring surgical revision. Within the first year after OLT, 6 patients deceased. Perioperative characteristics of patients and key laboratory parameters are depicted in Tables S1–S3 (SDC, http://links.lww.com/TP/B760).
Sdc-1 Concentrations in Effluents of the Human Liver Graft
In order to quantify the extent of glycocalyx degradation within the liver graft, we assessed Sdc-1 concentrations in the liver graft effluent. The median Sdc-1 concentrations in the effluent were 4720 [4374–5367] ng/mL, indicating that the glycocalyx is shed from the endothelium within the liver during ischemic graft preservation.
To evaluate whether the extent of glycocalyx degradation is in proportion to liver graft injury, we correlated effluent Sdc-1 concentrations with effluent concentrations of hepatic injury markers. Positive correlations were found between effluent Sdc-1 concentrations and effluent concentrations of the hepatic injury markers, including AP, AST, and lactate (Table 1). These findings suggest that the glycocalyx is degraded in proportion to liver graft injury. However, effluent Sdc-1 concentrations did not correlate with cold ischemia time or warm ischemia time (Table 1).
Sdc-1 Serum Concentrations in the Perioperative Phase
BL serum Sdc-1 concentrations in patients undergoing OLT were 191 ng/mL (IQR 103–303) and increased to 3293 ng/mL (IQR 2763–3941) at the end of OLT (P < 0.001). Serum Sdc-1 concentrations remained significantly elevated at day 1 (1017 ng/mL [IQR 633–2095], P < 0.001) and day 2 (810 ng/mL [IQR 450–1600], P = 0.01) when compared with BL, but did not differ from BL values on day 3 (630 ng/mL [IQR 340–1102], P = 0.18) after OLT. Of note, serum Sdc-1 concentration at day 1 after OLT correlated with warm ischemia time (Table 1).
Sdc-1 Concentrations and EAD After OLT
Effluent Sdc-1 concentrations were greater in patients who developed EAD compared with those who did not develop EAD (4720 ng/mL [4374–5133] vs 3838 ng/mL [3202–4240], P = 0.015; Figure 1). In addition, on postoperative day 1, patients who developed EAD displayed greater serum Sdc-1 concentrations than patients without EAD (1635 ng/mL [IQR 604–1974] vs 762 ng/mL [IQR 416–1247], P = 0.004; Table S1, SDC, http://links.lww.com/TP/B760).
Sdc-1 concentrations in the effluent and serum correlated with concentrations of serum AST and ALT (Table 1), suggesting association with EAD. When performing ROC curve analysis to assess whether glycocalyx shedding is associated with the development of EAD, we found a strong association between effluent Sdc-1 concentrations and EAD (AUC = 0.82, P = 0.017, accuracy 70%, NPV of 100%, and PPV of 36% for a cutoff value of >4031 ng/mL [Figure 2, Table S4, SDC, http://links.lww.com/TP/B760]). In addition, ROC curve analysis of serum Sdc-1 at day 1 after OLT revealed an AUC = 0.84 (P = 0.006, accuracy 74%, NPV of 100% and PPV of 30% for a cutoff value >971 ng/mL; Figure 2, Table S4, SDC, http://links.lww.com/TP/B760). Furthermore, logistic regression with EAD as the dependent variable (Table 2) showed that effluent Sdc-1 and serum Sdc-1 at day 1 were predictor variables for EAD in the univariate model. In the multivariate regression model, effluent Sdc-1 and serum Sdc-1 at day 1 remained independently associated with the development of EAD.
Sdc-1 Concentrations and 1-Year Graft and Patient Survival After OLT
All patients who developed graft dysfunction within the first year after OLT also deceased within this time frame. Effluent Sdc-1 concentrations as well as serum Sdc-1 concentrations did not differ among patients who survived the first year after OLT when compared with those who deceased within the first year after OLT (Table S2, SDC, http://links.lww.com/TP/B760).
Effluent Sdc-1 was not associated with 1-year survival in ROC analysis (AUC = 0.62, P = 0.44; Table S5, SDC, http://links.lww.com/TP/B760) and regression analysis (Table 3). Similarly, serum Sdc-1 was not associated with 1-year survival in ROC analysis (Table S5, SDC, http://links.lww.com/TP/B760) and regression analysis (Table 3). Only patients’ age was associated with 1-year survival in multivariate regression analysis (Table 3).
In this study, we investigated whether the endothelial glycocalyx is degraded within the human liver graft during ischemic preservation in the clinical setting of OLT and whether glycocalyx damage is related to the extent of liver graft injury. We report elevated Sdc-1 concentrations in the effluent of the liver graft, indicating that the glycocalyx is damaged during ischemic graft preservation. In addition, the extent of glycocalyx degradation within the human liver graft was related to the severity of liver graft injury. Moreover, effluent and serum Sdc-1 concentrations were greater in patients who developed EAD compared with those who did not develop EAD and associated with the development of EAD following OLT.
The endothelial glycocalyx, a surface layer of specialized proteoglycans and glycoproteins, is critically involved in the regulation of vascular function.23 The glycocalyx is responsible for maintaining vascular integrity and permeability and plays an important role in inflammatory, immunological, as well as coagulation processes.24 Degradation of the glycocalyx, which can be quantified by measuring Sdc-1 concentrations,8,25 has been associated with ischemia and reperfusion (I/R) injury in experimental and clinical studies.2,26-28 Although organ transplantation is unavoidably associated with I/R injury, only few data exist regarding endothelial glycocalyx damage in this clinical setting. In an experimental study, Rancan et al5 showed that lung transplantation induced glycocalyx degradation in pigs, which was subsequently associated with pulmonary edema and leucocyte infiltration within the graft. Results from a clinical study demonstrate extended glycocalyx damage within human kidney grafts during kidney transplantation.6 In addition, the authors observed that glycocalyx degradation was inversely correlated with the creatinine clearance after transplantation.6
We have previously demonstrated that serum Sdc-1 concentrations increase in the clinical setting of OLT.4 In this current study, we confirmed previous findings by measuring elevated systemic Sdc-1 concentrations in patients undergoing OLT. We further demonstrate that effluent Sdc-1 concentrations markedly exceed those in serum of recipients. These findings indicate that human liver grafts are a major source of Sdc-1, suggesting that the endothelial glycocalyx is damaged within the liver graft during ischemic preservation. Damage to the glycocalyx leads to cell edema, increased vascular permeability, activation of a pro-inflammatory response, and leukocyte adhesion,24,29 which can subsequently induce organ dysfunction.27,30 Therefore, extensive glycocalyx damage during graft preservation might contribute to hepatocellular injury within liver grafts. Hepatocellular injury within the liver graft is reflected by markers such as ALT, AST, AP, and lactate in the effluent.16 Thus, we correlated effluent Sdc-1 concentrations with effluent concentrations of these hepatic injury markers. As effluent concentrations of the glycocalyx damage marker Sdc-1 positively correlated with those of AST, AP, and lactate, our findings suggest that the extent of glycocalyx damage within liver grafts is proportional to the extent of hepatocellular injury.
Severe graft injury negatively impacts early postoperative graft function.9-11 Thus, our finding that glycocalyx damage is proportional to the extent of graft injury is supported by the fact that effluent Sdc-1 concentrations of patients who developed EAD were significantly greater than those of patients with normal graft function after OLT. Concordantly, systemic Sdc-1 concentrations were greater in patients who developed EAD than in patients without EAD. The increase of systemic Sdc-1 concentrations could be explained by other factors such as as surgical trauma, hypovolemia, perioperative fluid administration, or the use of blood products.8,25,27,31 However, the fact that both effluent and systemic Sdc-1 concentrations were greater in EAD patients than in non-EAD patients suggests that EAD patients might have received grafts with more pronounced glycocalyx damage.
Due to limited donor organ availability, transplantation centers increasingly consider extended criteria donors, resulting in collection of marginal grafts.32,33 Transplantation of a graft with marginal quality negatively impacts outcome,34,35 making direct assessment of graft quality before transplantation clinically important. Analyzing markers within the liver graft effluent and correlating them with early postoperative graft function might allow for the preoperative assessment of graft quality. Recently, Verhelst et al18 detected a strong association between effluent concentrations of protein-linked-N-glycans and the development of primary nonfunction. The authors suggested that the marker might be used to assess graft quality before transplantation.18 Similarly, Selten et al17 demonstrated that effluent concentrations of hepatocyte-derived microRNAs correlated with EAD and proposed that effluent mircoRNA concentrations are a promising marker indicating hepatocyte injury during preservation. As we observed increased glycocalyx damage within liver grafts of patients who developed EAD after OLT, we also assessed whether effluent Sdc-1 concentrations might be a marker for the development of EAD. ROC and regression analyses revealed that effluent Sdc-1 concentrations could indeed differentiate between patients developing EAD and those who did not develop EAD after OLT. In addition, serum Sdc-1 concentrations on day 1 after OLT were associated with the development of EAD. Taken together, in accordance to aforementioned effluent markers, Sdc-1 concentrations might also mirror graft quality in the clinical setting of OLT.
The diagnosis of EAD by currently used criteria is still issue of debate.9,15 However, elevated serum transaminases within the first week after OLT are not specific for EAD, but also reflect severe I/R injury, which often resolves within the first days after transplantation. Thus, markers predicting patients’ long-term outcome after OLT are probably of greater clinical interest. Recently, the liver transplant group of the Hospital de Clinicas of Porto Alegre in Brazil demonstrated that serum factor V is a promising marker for predicting outcome after OLT. Serum factor V was shown to predict graft dysfunction and identified as marker for predicting early graft loss and patient survival.36,37 Furthermore, Rostved et al showed that serum concentrations of hyaluronic acid, another component of the endothelial glycocalyx, independently predicted 1-year graft loss.38 Concordantly, we analyzed the association between Sdc-1 and 1-year graft survival and 1-year patient survival. In contrast to aforementioned studies, we did not observe a significant association between effluent Sdc-1 and 1-year graft survival. Similarly, we found no association between serum Sdc-1 and 1-year graft and 1-year patient survival. However, these missing associations between Sdc-1 concentrations and 1-graft survival or 1-patient survival have to be interpreted with caution. Our study was not designed nor powered to assess long-term outcomes such as 1-graft survival or 1-patient survival. In particular, within the first year after OLT only 4 patients lost their grafts and only 6 patients deceased.
Our study has further limitations that bear mentioning. We are not able to underline our clinical EAD diagnosis with histological findings, and we are aware that the sample size is a major limitation of our study. In particular, EAD was diagnosed only in 7 patients. It is therefore important to note that our findings concerning the prediction of EAD are derived from a small sample size and need to be validated in larger, independent cohorts. Furthermore, it appears reasonable that one single parameter cannot be expected to provide accurate predictive values in such a complex disease like EAD. An interplay between Sdc-1 and other promising predictive markers such as serum factor V36,37 and microRNAs in the effluent17 could contribute to a reliable prediction of EAD or 1-year outcome. Finally, in contrast to data from current literature, we detected no association with cold ischemia time. These contradictory findings might be explained by the fact that prolonged cold ischemia time over 10 hours has been associated with EAD,9,10,39 but none of our grafts was exposed to cold ischemia time over 10 hours.
Despite these limitations, the strength of our study is the prospective sampling and a well-characterized study cohort facilitating the hypothesis-driven analysis of Sdc-1 in various types of samples, including donor-graft effluent. This enabled us to demonstrate that assessing glycocalyx damage in the liver graft effluent and recipients’ serum might be a promising approach aiming to predict EAD after OLT. Furthermore, our findings are novel and could contribute to the early identification of EAD or even grafts with marginal quality. However, further investigations of the pathophysiology of I/R injury and glycocalyx degradation in OLT are needed to guide the development of interventions to prevent EAD.
In conclusion, our data suggest that the extent of glycocalyx shedding within the liver graft correlates with hepatocellular injury of the graft in human OLT. Moreover, recipients of livers grafts with greater glycocalyx shedding might be at greater risk to develop EAD after OLT.
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