Sirtuin-1 expression and activity is diminished in aged liver grafts

The cellular mechanisms underlying impaired function of aged liver grafts have not been fully elucidated, but mitochondrial dysfunction appears to be contributory. Sirtuin1 has been identified as a key mediator of mitochondrial recovery following ischemia–reperfusion injury. The purpose of this study was to determine whether differences exist in sirtuin-1 expression/activity in old vs. young liver grafts and to determine correlations with mitochondrial function, graft metabolic function, and graft injury. Old and young rat liver grafts (N = 7 per group) were exposed to 12 h of static cold storage (SCS), followed by a 2 h period of graft reperfusion ex vivo. Sirtuin1 expression and activity, mitochondrial function, graft metabolic function, and graft injury were compared. Sirtuin1 expression is upregulated in young, but not old, liver grafts in response to cold storage and reperfusion. This is associated with diminished tissue ATP, antioxidant defense, and graft metabolic function in old liver grafts. There was no evidence of increased inflammation or histologic injury in old grafts. Sirtuin1 expression is diminished in old liver grafts and correlates with mitochondrial and metabolic function. The sirtuin pathway may represent a target for intervention to enhance the function of aged liver grafts.

Study design. The study consisted of two experimental groups in which liver grafts were exposed to 12 h of cold storage followed by 2 h of ex vivo reperfusion: Old Lewis rats (26 months, N = 7) vs. young Lewis rats (2.5 months, N = 7). Three control groups consisting of untreated livers were also tested: old ACI rats (28 months, N = 6), young ACI rats (2.5 months, N = 6), and young Lewis rats (2.5 months, N = 6) (Fig. 1). Age and weights of the animals are shown in Table 1.

Experimental procedures
Liver procurement. As we previously described 21 , all surgeries were performed under cone mask anesthesia with continuous 5% isoflurane (Isothesia, Henry Schein Animal Health, Melville, NY) for induction, and 2-3% isoflurane during the procedure, with 2 L/minute oxygen flow. The abdominal cavity was opened by a midline and transverse incision. A stent fashioned from a 24-gauge angiocatheter (BD Insyte autoguard Becton Dickinson, Franklin Lakes, NJ) was inserted into the common bile duct and secured. The proper hepatic artery and gastrosplenic and duodenopancreatic branches of the portal vein were isolated and divided. Heparin (1 IU/g bodyweight, Fresenius Kai, Lake Zurich, IL) was injected through the infrahepatic vena cava with a 30-gauge needle. Then, 5 min later, the portal vein was cannulated with a perfusion cannula (Harvard Apparatus, Holliston, MA), and the liver gently flushed with 40 mL of cold University of Wisconsin (UW) solution (Bridge of Life, Columbia, SC). The liver was then explanted and weighed. www.nature.com/scientificreports/ Static cold storage. As we previously described 21 , for static cold storage (SCS), grafts were immersed in 50 mL UW solution (Bridge of Life) at 4 °C for 12 h. At different time points, livers were flushed with 10 mL UW solution. 1.5 mL flush solution was centrifuged at 2,500 rpm at 4 °C for 15 min to remove cellular debris. The supernatant was transferred to 1-mL Corning cryogenic vials immediately after centrifugation, frozen with liquid nitrogen and stored at − 80 °C for subsequent analysis.
Ex vivo graft reperfusion. As we previously described 21 , after cold storage preservation, grafts were transferred to the perfusion circuit and reperfused ex-vivo for 2 h. The reperfusion was performed in a recirculating system consisting of a perfusate reservoir, an autoclavable organ chamber (Type 834/10), an oxygenator, a peristaltic pump and a bubble trap (Hugo Sachs Elektronik, March-Hugstetten, Germany). The chamber was enclosed in a conditioning system, which allows precise regulation and control of the temperature (Optima T100, Grant instruments, Beaver Falls, PA). Perfusion media consisted of 100 mL oxygenated (95% O 2 and 5% CO 2 ; Airgas, Durham, NC) Krebs-Henseleit buffer (Sigma Aldrich, St. Louis, MO) supplemented with 5% bovine serum albumin (Hyclone, GE Healthcare Life Sciences, South Logan, UT), and 100,000 U/L penicillin, 100 mg/L streptomycin (Gibco-Life Technologies, Grand Island, NY). Perfusate flow rate was 3 mL/minute/gliver, and temperature was maintained at 37 °C. Before reperfusion, the liver was flushed with 20 mL 0.9% normal saline at room temperature and allowed to equilibrate for 15 min to simulate the performance of vascular anastomoses (equilibration phase).

Analytical methods
Intrahepatic vascular resistance. As we previously described 21 , the intrahepatic vascular resistance was calculated as portal vein pressure (mmHg)/perfusate flow rate (mL/minute). Vascular resistance ratio was calculated as the vascular resistance at a specific time point divided by the initial vascular resistance. During reperfusion, portal vein pressure was measured continuously (Flow pressure transducer P75, HSE amplifier module TAM-D; Hugo Sachs Elektronik). LabChart Pro software (AD Instruments, Colorado Springs, CO) was used to display and record flow rates and portal vein pressure.
Blood gas analysis. As we previously described 21 , perfusate samples were drawn from the portal venous inflow and from the suprahepatic caval outflow at different time points for blood gas analysis using a point-ofcare device (iSTAT, CG4 + cartridges, Abbott Point of Care Inc., Abbott Park, IL). Measurements included lactate concentration and acid-base parameters (pH, HCO 3 -, base excess, pO2, and pCO2).
Calculation of oxygen consumption rate. As we previously described 21 , the oxygen consumption rate during reperfusion was calculated using the following equation: Oxygen consumption rate (μL/minute/gliver) = perfusate flow x S x (P i O 2 -P o O 2 )/g-liver. In this equation, P i O 2 represents the partial pressure of oxygen of the inflow (portal vein), P o O 2 represents the partial pressure of oxygen of the outflow (suprahepatic vena cava), and S represents the solubility constant of oxygen in water at 37 °C (0.031 μL/mL/mmHg).
Bile processing and analysis. As we previously described 21 , bile was collected throughout reperfusion and volume was determined hourly. Bile was transferred to 1-mL Corning cryogenic vials (VWR, Atlanta, GA), frozen with liquid nitrogen and stored at -80 °C for subsequent analysis. Biliary lactate dehydrogenase (LDH) was determined using ELISA (LifeSpan BioSciences, Seattle, WA, USA), and biliary glucose was analyzed with the iSTAT point-of-care device (Chem8 + cartridges; Abbott Point of Care).
Perfusate analysis. As we previously described 21 , at different time points, 2 mL of perfusate was collected and centrifuged at 2,500 rpm at 4 °C for 15 min to remove cellular debris. The supernatant was transferred to 1-mL Corning cryogenic vials immediately after centrifugation, frozen with liquid nitrogen, and stored at -80 °C for subsequent analysis.
Liver damage parameters. Alanine aminotransferase (ALT) in perfusate was measured using a Piccolo Xpress Chemistry Analyzer (Abaxis, Union City, CA). LDH was determined using ELISA (LifeSpan BioSciences). Toll-like receptor activation. As we previously described 21 , TLR activation was measured using TLR reporter cell lines human embryonic kidney (HEK)-hTLR3, HEK-hTLR4, and HEK-hTLR9 cells (InvivoGen, San Diego, CA), stably expressing a nuclear factor kappa B (NF-κB)/activator protein 1-inducible secreted embryonic alkaline phosphatase (SEAP). All cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere with 5% CO2. Perfusates were diluted to 15% (vol/vol) in growth media and incubated with TLR reporter cells in flat-bottom 96-well plates for 16 h. SEAP levels were then determined by colorimetric assay using QUANTIBlue assay (InvivoGen). Polyinosinic: polycytidylic acid (5 μg/mL, InvivoGen), lipopolysaccharide (20 ng/mL, Sigma-Aldrich), and CpG (1 mM, Invi-voGen) were used as TLR3, TLR4, and TLR9 stimulator controls, respectively. Culture media from stimulated HEK-null cell lines (InvivoGen) were subtracted from experimental samples to account for endogenous levels of alkaline phosphatase. Liver tissue processing. Liver parenchyma was partitioned in three ways for assays. A portion of tissue was flash-frozen in liquid nitrogen and stored at − 80 °C for genomic DNA isolation, analysis of ATP concentration, ADP/ATP ratio, and sirtuin1 activity. Another portion of tissue was immersed in RNAlater Solution (Thermo Fisher Scientific, Waltham, MA, USA) and frozen at − 80 °C for later RNA isolation. A final portion of tissue was fixed in 10% formalin for histologic assessment.

Analysis of ATP concentration and ADP/ATP ratio.
Histology. Liver tissue was fixed in 10% formalin and paraffin embedded. Sections were obtained from the medial left lateral lobe. Paraffin sections were stained with hematoxylin and eosin (H&E) for morphologic observation. Severity of histological damage was blindly scored by Suzuki criteria after H&E staining. Sinusoidal congestion, hepatocyte necrosis, and ballooning degeneration were graded from 0 to 4 points, and the final score is the sum of the grades for each item as previously described 25 . Immunohistochemical (IHC) staining for NRF-1 (Abcam) was used to demonstrate the expression of NRF-1 in the cells. Apoptotic cells were identified by terminal dUTP nick-end labeling of fragmented DNA assay (TUNEL) (Roche, Mannheim, Germany). Whole slide digital images were captured by the Aperio AT Turbo digital slide scanner system (Leica Biosystems, Vista, CA). Quantitative immunohistochemical analysis was performed using Aperio Imagescope digital pathology software (Leica Biosystems). NRF-1 expression and TUNEL stain level were analyzed using an optimized positive pixel algorithm to obtain a percent pixel positivity of in the measured areas.  www.nature.com/scientificreports/

Results
Sirtuin1 expression is upregulated in young, but not old, liver grafts in response to cold storage and reperfusion. Sirtuin1 mRNA expression was significantly upregulated in young liver grafts in response to ischemia-reperfusion, while this was not observed for old grafts (Fig. 2a). Similarly, sirtuin1 protein levels were significantly increased in young liver grafts in comparison to both old liver grafts and untreated control liver tissue (Fig. 2b). Sirtuin1 enzyme activity was diminished in old liver grafts compared to control young liver tissue, which was not observed for young grafts (Fig. 2c).
Tissue ATP is lower in old liver grafts despite similar mitochondrial mass. Mitochondrial function was impaired in old liver grafts after reperfusion, as evidenced by lower tissue ATP levels and an elevated ADP: ATP ratio (Fig. 3a, b). Mitochondrial mass was lower in untreated old vs. young livers at baseline (Fig. 3c). However, in the experimental groups, there were no significant differences in mitochondrial mass observed for young and old liver grafts following reperfusion.

Antioxidant gene expression is impaired in old liver grafts.
Given the effect of age on sirtuin1 expression and the key role played by sirtuin1 in activating antioxidant defense mechanisms 19,20 , gene expression of antioxidant enzymes was compared in old vs. young liver grafts. Expression of superoxide dismutase-1 Figure 4. Antioxidant and cytoprotective gene expression. Tissue samples from old and young liver grafts following cold storage and reperfusion were assessed to determine the induction of antioxidant and cytoprotective genes. a Expression of superoxide dismutase-1 (SOD-1), the cytosolic form, is significantly lower in old liver grafts following cold storage and reperfusion. b SOD-2, the mitochondrial form, is significantly less in old untreated controls compared to young controls, indicating reduced expression at baseline. Following reperfusion, young liver grafts demonstrate significantly higher expression of SOD-2 while old liver grafts do not. c Heme oxygenase-1 (HMOX-1) expression was not significantly different between groups. Data shown as mean ± SEM, N = 4-6 per group, *p < 0.05, **p < 0.01. www.nature.com/scientificreports/ (SOD1), a cytosolic enzyme that acts on the superoxide anion, was significantly lower in old liver grafts in comparison to both young liver grafts and control liver tissue (Fig. 4a). Gene expression of SOD2, the mitochondrial form, was significantly upregulated in young, but not old, liver grafts in comparison to control liver tissue. SOD2 expression was lower in untreated old vs. young livers at baseline (Fig. 4b). Gene expression of heme-oxygenase-1, a cytoprotective enzyme involved in liver I-R, was not significantly different between groups (Fig. 4c).
Mitochondrial quality control pathways are impacted by cold storage and reperfusion. Mitochondrial biogenesis and mitophagy are two key pathways involved in mitochondrial quality control 26 . In the biogenesis pathway, sirtuin1 functions as an activator of PGC1α, a key transcription factor that coordinates the nuclear and mitochondrial response 27 . To determine the impact of sirtuin1 on this pathway, levels of PGC1α were measured in graft tissue. In both old and young liver grafts, levels of PGC1α were increased relative to control liver tissue, indicating the activation of mitochondrial biogenesis pathways (Fig. 5a). Interestingly, expression of a downstream transcription factor, NRF-1, was significantly reduced for both old and young liver grafts following ischemia-reperfusion (Fig. 5b). To assess changes in mitophagy, gene expression of Parkin was compared for graft and control liver tissue. Parkin is responsible for the ubiquitylation of outer mitochondrial mem- Figure 5. Mitochondrial quality control pathways. Tissue samples from old and young liver grafts following cold storage and reperfusion were assessed to determine levels of key regulators of mitochondrial biogenesis and mitophagy. a PGC-1α, an early activator of the mitochondrial biogenesis pathway, is greater in both old and young liver grafts following cold storage and reperfusion. b Gene expression of Parkin, an initiator of mitophagy, is lower in both old and young liver grafts following cold storage and reperfusion. c Nuclear expression of NRF-1, a downstream transcription factor in the mitochondrial biogenesis pathway, is significantly lower following cold storage and reperfusion, reflecting the lag between PGC1α activity and downstream NRF-1 expression. Data shown as mean ± SEM, N = 4-6 per group, *p < 0.05, **p < 0.01, ***p < 0.001. www.nature.com/scientificreports/ brane proteins and initiation of mitophagy 28 . Interestingly, in both old and young livers grafts, Parkin expression was significantly decreased following cold storage and reperfusion (Fig. 5b).
Transmission electron microscopy (TEM) demonstrates morphologic differences between old and young mitochondria. TEM was performed to assess whether any morphologic differences exist between old and young mitochondria exposed to ischemia-reperfusion. TEM analysis demonstrated swelling of cristae in old mitochondria. More prominent signs of mitochondrial damage, including organelle swelling, were absent (Fig. 6).
Old liver grafts sustain greater injury during cold storage, reflected by DAMP release. To assess graft injury during cold storage, levels of exDNA and HMGB1, two DAMPs associated with ischemic liver injury 29,30 , were measured at 1 h, 3 h, 6 h, and 12 h (Fig. 7). Both ExDNA and HMGB1 levels were significantly  Old liver grafts demonstrate impaired metabolic function and increased injury during reperfusion. Impaired metabolic function of older grafts was demonstrated by several parameters, including lower bile production, reduced oxygen consumption, and higher perfusate lactate in comparison to young liver grafts (Fig. 8a-c). Old grafts also demonstrated impaired gluconeogenesis and glycogenolysis, evidenced by significantly lower glucose release into the perfusate (Fig. 8d). In addition, old liver grafts demonstrated increased injury during reperfusion, evidenced by higher vascular resistance (Fig. 8e), higher perfusate ALT (Fig. 8g), and higher perfusate LDH at 30 min (Fig. 8h). Other markers of injury were not significantly different between old and young liver grafts, including vascular resistance ratio (Fig. 8f), biliary LDH (Fig. 8i), perfusate exDNA (Fig. 8j), and perfusate HMGB1 (Fig. 8k). ExDNA levels were markedly increased (fivefold or more) during reperfusion compared to the end of SCS, while HMGB1 levels were similar between the reperfusion and SCS phases ( Fig. 7 and Fig. 8j, k). The oxygen consumption rate of old liver grafts was significantly lower during the early reperfusion period, reflecting a lower graft metabolic rate. c Lactate levels in the perfusate were significantly higher for old liver grafts during the early reperfusion period, indicating impaired metabolic function. d Glucose release into the perfusate was significantly lower for old grafts, indicating impaired gluconeogenesis and glycogenolysis. e Vascular resistance during reperfusion was higher for old grafts, indicative of microvascular injury. f However, the vascular resistance ratio was similar between old and young liver grafts over the course of reperfusion. Markers of hepatocellular injury including g ALT and h LDH were significantly higher for old liver grafts in the early reperfusion period. i Biliary LDH levels, indicative of injury to biliary epithelium, were not significantly different between old and young grafts. Levels of j extracellular DNA (exDNA) and k HMGB1 released into perfusate were not significantly different between groups. Data shown as mean ± SEM, N = 7 per group, *p < 0.05, **p < 0.01, ***p < 0.001. www.nature.com/scientificreports/

Release of inflammatory molecules and induction of inflammatory genes does not differ between old and young liver grafts.
To assess the proinflammatory milieu associated with reperfusion of old and young grafts, the release of TLR agonists and inflammatory cytokines was measured during reperfusion. There were no significant differences between old and young liver grafts in release of TLR-stimulating molecules (Fig. 9a-c). Similarly, there were no significant differences in the release of IL-1α or TNFα during reperfusion (Fig. 9d, e). Following reperfusion, gene expression of the inflammatory cytokines TNFα and IL-6 was significantly upregulated in both old and young liver grafts, but no significant differences existed based on age (Fig. 9f, g).
Histologic injury and degree of apoptosis do not significantly differ between old and young liver grafts. Following graft reperfusion, histologic analysis was performed to assess degree of graft injury.
Both old and young liver grafts demonstrated significantly greater histologic injury compared to control liver Figure 9. Inflammatory profile of old and young liver grafts. Perfusate samples were collected during reperfusion and Toll-like receptor (TLR) activation was measured by incubating perfusate with TLR reporter cell lines. Levels of inflammatory cytokines released into perfusate were measured by cytometric bead array. Tissue samples of liver grafts at the end of reperfusion and untreated control tissue were assessed for inflammatory gene expression. a TLR3, b TLR4, and c TLR9 activation by perfusate from old versus young liver grafts was not significantly different, indicating that liver grafts release comparable levels of inflammatory mediators during reperfusion. Similarly, levels of inflammatory cytokines d IL-1α and e TNF-α released into the perfusate were not significantly different between old versus young liver grafts. Inflammatory gene expression of f TNF-α and g IL-6 were not significantly different between old and young liver grafts, but significantly higher than untreated control livers. Data shown as mean ± SEM, N = 4-7 per group, *p < 0.05, **p < 0.01, ***p < 0.001. www.nature.com/scientificreports/ tissue as assessed by Suzuki score (Fig. 10a), but there was no significant difference based on age. To assess the degree of apoptosis in the tissues, TUNEL staining was performed. Both old and young liver grafts demonstrated significantly greater apoptosis compared to control liver tissue, but again no significant differences were observed based on age (Fig. 10b).

Discussion
The underlying cellular mechanisms contributing to increased susceptibility of old liver grafts to I-R injury have not been fully elucidated. Recent mechanistic studies indicate that diminished sirtuin-1 activity in aged hepatocytes contributes to an impaired mitochondrial response to I-R injury in vitro 16 , but the relevance of this pathway in liver transplantation remains unknown. In this study, we demonstrate that old liver grafts exhibit decreased expression and activity of sirtuin-1 (Fig. 2), which correlates with impaired mitochondrial function (Fig. 3), and ultimately impaired graft metabolic function (Fig. 8). The findings in our study are consistent with a recent study by Nakamura and colleagues who demonstrated that increased sirtuin-1 protein expression in Figure 10. Histologic injury and apoptosis following graft reperfusion. Tissue samples from old and young liver grafts following cold storage and reperfusion were assessed to determine histologic injury by H&E and degree of apoptosis by TUNEL staining. a H&E staining demonstrates significantly increased hepatocyte vacuolization, congestion, and necrosis at the end of reperfusion in both old and young grafts, reflected in the Suzuki score. b TUNEL staining demonstrates significantly more apoptosis in liver grafts compared to control tissue, but no significant difference between old and young liver grafts. Data are shown as mean ± SEM, N = 4-6 per group, *p < 0.05, ***p < 0.001. www.nature.com/scientificreports/ post-reperfusion liver biopsies was associated with reduced early allograft dysfunction and enhanced graft survival in human liver transplants 31 . Due to limited sample size, however, Nakamura was unable to demonstrate an association between donor age and sirtuin-1 expression. From a mechanistic perspective, the effects of sirtuin1 on mitochondrial function are mediated in part by activation of antioxidant defense mechanisms 19,20 and mitochondrial biogenesis pathways 27 . In this study, we demonstrate a correlation between diminished sirtuin1 in old liver grafts and impaired antioxidant gene expression (Fig. 4), but did not observe any impairment in mitochondrial biogenesis activity (Fig. 5). The latter observation may be due to the relatively early time point at which we made our assessment (2 h post reperfusion). Importantly, the diminished function of old grafts does not appear to be driven by greater inflammation (Fig. 9) or increased histologic injury (Fig. 10). Our study also confirms the increased susceptibility of old liver grafts to cold storage preservation injury. Release of extracellular DNA and HMGB1, two DAMPs indicative of cell death, were significantly elevated in the cold storage preservation solution of old liver grafts (Fig. 7). This finding is consistent with clinical reports demonstrating an association between shorter cold ischemic time and improved outcomes with older donor grafts 2 . This concept also supports the potential use of normothermic machine perfusion for the preservation of old grafts to reduce exposure to cold ischemia.
There are some important limitations of this study that should be recognized. Due to the large size of the old liver grafts, it was not technically feasible to perform orthotopic liver transplants into younger recipients. As such, graft reperfusion was performed by ex vivo reperfusion with an acellular perfusate (Krebs-Henseleit buffer). We selected this model because it is a standardized approach for ex vivo liver reperfusion experiments in the literature [32][33][34][35][36][37] , but acknowledge that liver transplantation is the optimal reperfusion model. Second, we assessed mitochondrial function indirectly by measuring ATP and ADP levels in the tissue. While this is a standard approach in ischemia-reperfusion models 14,17 , alternative techniques to directly measure mitochondrial function such as high resolution respirometry may provide more precision. Finally, due to the limited availability of old rats, we included 2 strains (Lewis and ACI), with the ACI strain serving as the untreated control. The high level of concordance observed between these 2 strains in the young cohort is reassuring that this comparison is valid.
In conclusion, this study supports the emerging importance of sirtuin-1 in liver transplantation and highlights age-related differences that may contribute to the discrepancy in outcomes between young and old liver grafts. The delivery of pharmacologic activators of sirtuin-1 during machine perfusion may represent a therapeutic strategy to improve the function of old liver grafts.

Data availability
The datasets generated during the current study are available from the corresponding author on reasonable request.