IGL-1 Solution Research Articles

Bortezomib enhances fatty liver preservation in Institut George Lopez-1 solution through adenosine monophosphate activated protein kinase and Akt/mTOR pathways

The aim of this study is to investigate the protective mechanisms induced by bortezomib added to Institut George Lopez (IGL)-1 preservation solution to protect steatotic livers against cold ischaemia reperfusion injury and to examine whether these mechanisms occur through the activation of adenosine monophosphate activated protein kinase (AMPK), Akt/mTOR pathways. Steatotic livers from obese rats were preserved for 24 h (at 4 °C) in IGL-1 solution with or without bortezomib (100 nM) or pretreated with AMPK inhibitor adenine 9-α-D-arabinofuranoside and preserved in IGL-1 + bortezomib. Livers were then perfused for 2 h at 37 °C. Liver injury (alanine aminotransferase/aspartate aminotransferase) and function (bile production and vascular resistance) were measured. Also, Akt/mTOR, phosphorylated AMPK (pAMPK) and apoptosis were determined by Western blot analyses. Bortezomib addition to IGL-1 solution significantly reduced steatotic liver injury, improved graft function and decreased liver apoptosis. These benefits were diminished by the pretreatment of obese rats with AMPK inhibitor Ara. Western blot analyses showed a significant increase in pAMPK after ischaemia and reperfusion. We also observed a significant phosphorylation of Akt in IGL-1 +bortezomib group that, in turn, induced the phosphorylation of mTOR and glycogen synthase kinase 3β. Bortezomib, at low and non toxic concentration, is a promising additive to IGL-1 solution for steatotic liver preservation. Its protective effect is due to the activation of AMPK and Akt/mTOR pathways.

AMPK involvement in endoplasmic reticulum stress and autophagy modulation after fatty liver graft preservation: a role for melatonin and trimetazidine cocktail

Ischemia/reperfusion injury (IRI) associated with liver transplantation plays an important role in the induction of graft injury. Prolonged cold storage remains a risk factor for liver graft outcome, especially when steatosis is present. Steatotic livers exhibit exacerbated endoplasmic reticulum (ER) stress that occurs in response to cold IRI. In addition, a defective liver autophagy correlates well with liver damage. Here, we evaluated the combined effect of melatonin and trimetazidine as additives to IGL-1 solution in the modulation of ER stress and autophagy in steatotic liver grafts through activation of AMPK. Steatotic livers were preserved for 24hr (4°C) in UW or IGL-1 solutions with or without MEL+TMZ and subjected to 2-hr reperfusion (37°C). We assessed hepatic injury (ALT and AST) and function (bile production). We evaluated ER stress (GRP78, PERK, and CHOP) and autophagy (beclin-1, ATG7, LC3B, and P62). Steatotic livers preserved in IGL-1+MEL+TMZ showed lower injury and better function as compared to those preserved in IGL-1 alone. IGL-1+MEL+TMZ induced a significant decrease in GRP78, pPERK, and CHOP activation after reperfusion. This was consistent with a major activation of autophagic parameters (beclin-1, ATG7, and LC3B) and AMPK phosphorylation. The inhibition of AMPK induced an increase in ER stress and a significant reduction in autophagy. These data confirm the close relationship between AMPK activation and ER stress and autophagy after cold IRI. The addition of melatonin and TMZ to IGL-1 solution improved steatotic liver graft preservation through AMPK activation, which reduces ER stress and increases autophagy.

Chronic renoprotective effect of pulsatile perfusion machine RM3 and IGL-1 solution in a preclinical kidney transplantation model

BackgroundMachine perfusion (MP) of kidney graft provides benefits against preservation injury, however decreased graft quality requires optimization of the method. We examined the chronic benefits of MP on kidney grafts and the potential improvements provided by IGL-1 solution.MethodWe used an established autotransplantation pig kidney model to study the effects of MP against the deleterious effects of warm ischemia (WI: 60 minutes) followed by 22 hours of cold ischemia in MP or static cold storage (CS) followed by autotransplantation. MPS and IGL-1 solutions were compared.ResultsAnimal survival was higher in MPS-MP and both IGL groups. Creatinine measurement did not discriminate between the groups, however MPS-MP and both IGL groups showed decreased proteinuria. Chronic fibrosis level was equivalent between the groups. RTqPCR and immunohistofluorescent evaluation showed that MP and IGL-1 provided some protection against epithelial to mesenchymal transition and chronic lesions. IGL-1 was protective with both MP and CS, particularly against chronic inflammation, with only small differences between the groups.ConclusionIGL-1 used in either machine or static preservation offers similar levels of protection than standard MP. The compatibility of IGL-1 with both machine perfusion and static storage could represent an advantage for clinical teams when choosing the correct solution to use for multi-organ collection. The path towards improving machine perfusion, and organ quality, may involve the optimization of the solution and the correct use of colloids.

Renoprotective effect of pulsatile perfusion machine RM3: pathophysiological and kidney injury biomarker characterization in a preclinical model of autotransplanted pig

To assess the effect of machine perfusion (MP) on renal function recovery vs kidney preservation in static cold storage (CS), in a large animal preclinical model. To assess whether MP benefits are dependent on the preservation solution used. Using an established autotransplantation pig kidney model associated with a contralateral nephrectomy we studied the impact of MP against the deleterious effects of warm ischaemia (WI; 60 min), then 22 h of cold ischaemia using MP or static CS, followed by autotransplantation. We used Berzer MP solution (MPS), recommended for MP, and Institut Georges Lopez preservation solution (IGL-1), designed for CS. The pigs were divided into four study groups: MPS-CS: static CS with MPS (n = 7); MPS-MP: renal perfusion with MPS using the Waters Medical Systems (Rochester, MN, USA) RM3 pulsatile machine (n = 7); IGL-CS: static CS with IGL-1 solution (n = 7); IGL-MP: renal perfusion with IGL-1 solution (n = 7). The effect of ischaemia was determined using different variables: pig survival; plasma creatinine; proteinuria; oxidative stress; tubular sodium reabsorption rate; and tissue damage at 1 month. Pig survival was higher in MP and IGL groups compared to MPS-CS. Plasma creatinine levels did not differ among the groups, but proteinuria assay showed significant benefits for the MP vs static CS groups. Histological evaluation of kidney grafts showed more injury in the CS groups than in the MP groups. Urinary measurement of tubular enzyme activity differed substantially among the groups, highlighting the benefits of MP in maintaining brush border integrity. In our model reproducing the conditions of deceased after cardiac arrest donors we show that MP decreases the risk of renal dysfunction and preserves kidney parenchyma. A non-invasive urinary enzyme assay can provide valuable information on graft integrity. The preservation solution used is important as the wrong solution can decrease the benefits of MP.

How Institut Georges Lopez Preservation Solution Protects Nonsteatotic and Steatotic Livers Against Ischemia-Reperfusion Injury

The Institut Georges Lopez preservation solution (IGL-1) is a serum-free organ preservative that has been shown to protect steatotic livers against hepatic ischemia-reperfusion injury. Although several hypotheses have been proposed to explain the graft protection mechanisms induced by IGL-1 solution, they have not been fully investigated. This review assessed possible IGL-1 mechanisms responsible for the increased liver tolerance of ischemia-reperfusion injury with special emphasis on vasodilatator mediators such as nitric oxide, on oxidative stress prevention, on protection against mitochondrial damage, and finally on induction of cytoprotective factors.

Melatonin protects steatotic and nonsteatotic liver grafts against cold ischemia and reperfusion injury

Chronic organ-donor shortage has required the acceptance of steatotic livers for transplantation purposes despite the higher risk of graft dysfunction or nonfunction associated with the cold ischemia-reperfusion injury. This study evaluated the use of melatonin as an additive to Institute Georges Lopez (IGL-1) solution for protecting nonsteatotic and steatotic liver grafts against cold ischemia-reperfusion injury. In the current investigation, we used an ex vivo isolated perfused rat liver model. Steatotic and nonsteatotic livers were preserved for 24 hr (4°C) in University of Wisconsin or IGL-1 solutions with or without melatonin, as well as in University of Wisconsin solution alone. Thereafter, livers were subjected to 2-hr reperfusion (37°C). We assessed hepatic injury (transaminases) and function [bile production and sulfobromophthalein (BSP) clearance, vascular resistance], as well as other factors potentially implicated in the high vulnerability of steatotic livers against ischemia-reperfusion injury (oxidative stress and related inflammatory mediators including nitric oxide and cytokines). We also evaluated well-known cytoprotective factors as hemeoxygenase 1 (HO-1). Fatty livers preserved in IGL-1 solution enriched with melatonin showed lower transaminase levels and higher bile production and BSP clearance when compared to those obtained for livers maintained in IGL-1 solution alone. A significant diminution of vascular resistance was also observed when melatonin was added to the IGL-1 solution. The melatonin benefits correlated with the generation of nitric oxide (through constitutive e-NOS activation) and the prevention of oxidative stress and inflammatory cytokine release including tumor necrosis factor and adiponectin, respectively. The addition of melatonin to IGL-1 solution improved nonsteatotic and steatotic liver graft preservation, limiting their risk against cold ischemia-reperfusion injury.

Relevance of Epidermal Growth Factor to Improve Steatotic Liver Preservation in IGL-1 Solution

Aim Static preservation solution is critical for liver graft outcomes, especially when steatosis is present. Institut Georges Lopez (IGL)-1 solution protects fatty livers effectively against cold ischemia reperfusion injury. Its benefits are mediated by nitric oxide and prevention of oxidative stress. The supplementation of IGL-1 with epidermal growth factor (EGF) enhances steatotic graft preservation by increasing adenosine triphosphate content, thereby mitigating oxidative stress and mitochondrial damage. Methods After steatotic livers were preserved for 24 hours in IGL-1 solution with or without EGF supplements, they were perfused ex vivo for 2 hours at 37°C. The benefits of EGF were assessed by evidences of hepatic damage and function—transaminases, bile production, and flow rate—as well as by other factors presumably associated with the poor tolerance of fatty livers toward cold ischemia-reperfusion injury (IRI)—energy metabolism, mitochondrial damage, oxidative stress, eNOS activity and proinflammatory interleukin (IL) beta content. Results Steatotic livers preserved in IGL-1 solutions supplemented with EGF (10 μg/L) showed lower transaminase levels, greater bile production, and ameliorated flow rates when compared to IGL-1 alone. In addition, energy metabolism deterioration, mitochondrial damage, oxidative stress, and cytokine IL-1 beta release were prevented. Conclusion EGF addition to IGL-1 increased fatty liver graft preservation, thereby reducing steatotic liver damage against cold IRI.

Hypoxia inducible factor-1α accumulation in steatotic liver preservation: Role of nitric oxide

To examine the relevance of hypoxia inducible factor (HIF-1) and nitric oxide (NO) on the preservation of fatty liver against cold ischemia-reperfusion injury (IRI). We used an isolated perfused rat liver model and we evaluated HIF-1alpha in steatotic and non-steatotic livers preserved for 24 h at 4 degrees C in University of Wisconsin and IGL-1 solutions, and then subjected to 2 h of normothermic reperfusion. After normoxic reperfusion, liver enzymes, bile production, bromosulfophthalein clearance, as well as HIF-1alpha and NO [endothelial NO synthase (eNOS) activity and nitrites/nitrates] were also measured. Other factors associated with the higher susceptibility of steatotic livers to IRI, such as mitochondrial damage and vascular resistance were evaluated. A significant increase in HIF-1alpha was found in steatotic and non-steatotic livers preserved in IGL-1 after cold storage. Livers preserved in IGL-1 showed a significant attenuation of liver injury and improvement in liver function parameters. These benefits were enhanced by the addition of trimetazidine (an anti-ischemic drug), which induces NO and eNOS activation, to IGL-1 solution. In normoxic reperfusion, the presence of NO favors HIF-1alpha accumulation, promoting also the activation of other cytoprotective genes, such as heme-oxygenase-1. We found evidence for the role of the HIF-1alpha/NO system in fatty liver preservation, especially when IGL-1 solution is used.

IGL‐1 solution in kidney transplantation: first multi‐center study

IGL-1 solution is characterized by inversion of K+ and Na+ concentrations in the University Wisconsin (UW) solution and polyethylene glycol 35 (PEG 35) substitution for hydroxy ethyl starch. In this prospective study, 121 patients transplanted with kidneys preserved in IGL-1 solution were compared to 102 patients grafted with kidneys preserved in UW solution. Serum creatinine and creatinine clearance, delayed graft function (DGF) and rejection episodes, patient and graft survival were evaluated in the first post-transplant year. Groups were comparable regarding to donor and recipient characteristics. Median creatinine levels were significantly lower in IGL-1 group from day 6 to day 14 and it decreased more rapidly in the IGL-1 group (from day 4 to day 15: p < 0.05). Creatinine clearance values were usually higher in the IGL-1 group for the first 15 d. During the follow-up period serum creatinine concentrations were lower in IGL-1 group at one, three, six and 12 months after transplantation (p = 0.04; p = 0.06, p = 0.01 and p = 0.08, respectively) while creatinine clearance values were similar during the follow-up. No significant difference in DGF and rejection rates as well as in patient and graft survival was shown between the two groups. Kidneys preserved in IGL-1 solution showed to have the same function as kidneys preserved in UW solution.

In Situ Intestinal Ischemia‐Reperfusion Injury in the Pig: A Model Using the First Jejunal Artery for Flushing

We describe a new surgical technique of in situ intestinal ischemia-reperfusion injury in the pig, which includes transection of the small bowel, extrinsic autonomic denervation, lymphatic disruption, and finally in-situ cold ischemia of the graft by flushing through the first jejunal artery. Ten female pigs were used for the study. All neural and lymphatic connections to the jejunoileum were transected. The stripped superior mesenteric vessels remained as the only connections. The skeletonized mesenteric vessels were clamped and the superior mesenteric artery was cannulated through the first jejunal artery. The isolated jejunoileum was flushed with cold IGL-1 solution. A small incision on the superior mesenteric vein was made to allow outflow of the effluent. After the flushing process was complete, the small incision in the superior mesenteric vein was closed and the vascular clamps were removed. The proximal 70% of the graft was resected. The mean preoperative weight of the animals was 25.8 +/-7.6 kg. The mean duration of the operation was 242.0 +/- 28.6 min. The mean cold ischemia time was 47.6 +/- 3.9 min. All animals survived the procedure and were sacrificed at day 8. At sacrifice, there were no adhesions. The small bowel appeared normal. On intestinal histology, there were no significant changes between specimens obtained from the animal immediately at the end of cold flushing (T0), 2 h after reperfusion (T1), and at sacrifice (T2). This novel technique for intestinal ischemia-reperfusion injury in the pig provides an extremely useful model for experimental studies of immunological and cold ischemia-reperfusion injury of transplanted small bowels.

Preservation of steatotic livers in IGL-1 solution

A new Institut Georges Lopez (IGL-1) solution was used to preserve steatotic livers. Steatotic (obese [Ob]) and nonsteatotic (lean [Ln]) livers from Zücker rats (n = 16, 8 Ln and 8 Ob) were preserved for 24 hours at 4 degrees C in University of Wisconsin (UW) or IGL-1 solution, respectively, and then perfused ex vivo for 2 hours at 37 degrees C. Additionally, Ob and Ln livers (n = 16, 8 Ln and 8 Ob) were preserved in IGL-1 plus Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME). Hepatic injury and function (aminotransferases, bile production, bromosulfophthalein clearance), and factors potentially involved in the susceptibility of steatotic livers to ischemia-reperfusion injury, such as oxidative stress, mitochondrial damage, and vascular resistance, were studied. Nitric oxide (NO) production and constitutive and inducible NO synthase were also measured. Steatotic and nonsteatotic livers preserved in IGL-1 solution showed lower transaminases, malondialdehyde, glutamate dehydrogenase levels, and higher bile production than UW-solution-preserved livers. IGL-1 solution protected against oxidative stress, mitochondrial damage and the alterations in vascular resistance associated with cold ischemia-reperfusion. Thus, at the end of reperfusion period, aspartate aminotransferase levels in steatotic livers were 281 +/- 6 U/L in UW vs. 202 +/- 10 U/L in IGL-1 solution. Glutamate dehydrogenase was 463 +/- 75 U/L in UW vs. 111 +/- 4 U/L in IGL-1 solution, and oxidative stress was 3.0 +/- 0.1 nmol/mg prot in UW vs. 2.0 +/- 0.1 nmol/mg prot in IGL-1 solution. These beneficial effects of IGL-1 solution were abolished by the addition of L-NAME, which implicates NO in the benefits of IGL-1. In conclusion, IGL-1 solution provided steatotic livers with better protection against the deleterious effects of cold ischemia-reperfusion injury than did UW solution.

Evaluation of IGL-1 preservation solution using an orthotopic liver transplantation model

To compare, in a pig liver transplantation model, the protective effect of UW with that of IGL-1, a high-sodium preservation solution containing polyethylene glycol (PEG) as an oncotic supply. All livers were harvested and grafted orthotopically according to standard techniques. The livers were washed out and preserved for 7 h in IGL-1 (n = 6) or in UW solution (n = 7) at 4 degree centigrade. In a sham group (n = 4), the livers underwent a 60-min warm ischemia at 37 degree centigrade. The hepatocellular injury was assessed in organ preservation solution washed out from the graft at the end of ischemic storage (before revascularization), and in serum 2 h after reperfusion and daily for up to 6 d. Livers preserved in IGL-1 solution released markedly less AST than that preserved in the UW solution before and after revascularization (P < 0.05). Besides, the activity of creatine kinase-BB, a marker of sinusoidal lining cells injury, was higher in the UW group than in the IGL-1 group (P < 0.05). Histological results showed less necrotic regions in livers preserved in IGL-1 solution; however, no difference was observed for inflammation. IGL-1 liquid effectively protects parenchymal and non-parenchymal cells against preservation-reperfusion injuries.

A Comparison of Cold Storage Solutions for Pancreas Preservation Prior to Islet Isolation

Background Several solutions are used to preserve the pancreas prior to islet isolation. This study sought to assess whether the type of solution had an impact on the isolation outcome. Methods We reviewed data from 125 islet isolation procedures performed from January 2002 to January 2005. Pancreata were preserved in University of Wisconsin (UW) ( n = 101), Celsior (CS) ( n = 19), or IGL-1 ( n = 5) solutions. Islet isolation results and transplantation rates were compared between groups. Results UW, CS, and IGL-1 groups were similar according to donor’s age, weight, and body mass index. Weight of undigested pancreas was 20 ± 13.1, 21.4 ± 15.7, and 17.4 ± 8.7 g for UW, CS, and IGL-1, respectively ( P > .2). Final total number of IEQ was 267,000 ± 132,000, 277,000 ± 155,000, and 311,000 ± 163,000, respectively ( P > .4). Success rate (defined as >250,000 IEQ) was 55.5%, 52.9%, and 60% for UW, Celsior, and IGL-1 ( P > .9); the transplantation rate was 42.2% for UW, 36.8% for Celsior, and 80% for IGL-1 preservation ( P > .2). Conclusions In this preliminary study, UW, Celsior, and IGL-1 solutions demonstrated similar islet isolation results. The new IGL-1 solution appears promising.

Effect of IGL-1, a new preservation solution, on kidney grafts (a pre-clinical study)

Ischemia-reperfusion injury conditions short-term and long-term graft function. The effects of the inversion of K+ and Na+ concentrations and substitution with polyethylene glycol for hydroxyethyl starch in University of Wisconsin (K-UW) solution were evaluated in isolated perfused rat kidneys and in autotransplanted pig kidneys. In the rat model kidneys were cold-stored for 24 h in K-UW or Na-UW or Na-PEG UW solutions (IGL-1 solution). Fractional sodium reabsorption and glomerular filtration rate was better in kidneys preserved in Na-UW and IGL-1 solution than those preserved in K-UW solution. In the pig model the left kidney was harvested and preserved in K-UW or IGL-1 solution for 24 h and then transplanted. In the autotransplanted pig model, kidneys preserved in IGL-1 solution showed a better function and a significant reduction of MHC class II expression, cellular apoptosis and interstitial fibrosis. In conclusion, kidneys preserved in IGL-1 solution tolerated ischemia/reperfusion injury better than those preserved in K-UW solution.

Kidney preservation with IGL-1 solution: A preliminary report

The University of Wisconsin (UW) solution is the most commonly used preservation solution. However, a new preservation solution—IGL-1—contains an inversion of K and Na concentrations and substitution of polyethylene glycol for hydroxyethyl starch in the UW solution. The present study is the first clinical experience on the outcome of kidneys preserved in IGL-1 solution. From June 2003 to June 2004, 119 cadaveric kidneys were retrieved and stored in IGL-1 solutions; among the 119 organs, this study includes 37 IGL-1-preserved kidneys that were locally transplanted versus 33 kidneys stored in University of Wisconsin (UW) solution that were also locally transplanted. The groups were comparable with regard to donor and recipient characteristics. Renal function outcome was evaluated by comparing delayed graft function (DGF) rates, the evolution of serum creatinine, daily urine output, and creatinine clearance. Biopsies were performed after reperfusion to evaluate apoptosis. The incidence of DGF was 5.71% among IGL-1 kidneys and 13.79% among UW kidneys. Creatinine values were significantly lower among the IGL-1 group from 2 to 14 days postoperative and at 1 month. Daily urinary output did not show any significant differences between the two groups. IGL-1 kidneys had a superior creatinine clearance during the first 15 postoperative days compared to UW kidneys. Kidneys preserved in IGL-1 solution showed fewer apoptotic cells compared to kidneys preserved in UW solution. This preliminary report suggests a superiority of IGL-1 for the immediate outcome of transplanted kidneys.

KIDNEY PRESERVATION WITH IGL-1 SOLUTION: A PRELIMINARY REPORT

P40 Aims: Although the University of Wisconsin (UW) solution continues to be the most commonly used preservation solution in multiorgan cadaveric donors, the new solution IGL-1 has been proposed for kidney, liver and pancreas preservation. Its peculiar characteristics are the inversion of K and Na concentration and the substitution with polyethylene glycol for hydroxyethyl starch in UW solution. In the present study we report the first clinical experience started in June 2003 on the outcome of the kidneys preserved in IGL-1 solution in relation to those preserved in UW solution. Methods: We collected the data from 52 cadaveric kidneys preserved in IGL-1 solution and 33 with UW solution. Renal function outcome was evaluated by delayed graft function (DGF) rate, daily urinary output, evolution of serum and urinary creatinine. All the values are expressed as median numbers and statistical studies included a multivariate analysis concerning donor and recipient characteristics and a Mann-Whitney test to assess differences between the groups. Results: The 52 kidneys preserved in IGL-1 solution originated from donors aged from 19 to 70 years (44 yrs), 29 males and 23 females, which were transplanted in recipients aged from 24 to 67 years (48.5 yrs), 32 males and 20 females; cold ischemia time was 16.08 hrs and in 10.4% of the cases it was more than 24 hrs. The 33 kidneys preserved in UW solution originated from donors aged from 16 to 69 years (43.5 yrs), 21 males and 12 females, which were transplanted in recipients aged from 25 to 70 years (48 yrs), 19 males and 14 females; cold ischemia time was 15.83 hrs and in 15.15% of the cases it was more than 24 hrs. The daily evolution of serum creatinine and urinary output were similar between the two groups although a lower Δ-creatinine at day 0-day 1 and a lower creatinine value at day 2 in IGL-1 group. No significant difference in serum creatinine values was shown at 1, 3, 6 months post-transplantation between the two groups. Although there was not a significant difference in Δ urinary creatinine between the two groups, daily urinary creatinine were significantly higher at days 6, 7, 9, 11 and 30 in IGL-1 group. The incidence of DGF, although not statistically significant, was lower in IGL-1 kidneys (7.5%) compared to UW-preserved kidneys (12.1%). Kidney biopsies were obtained before engraftment into the recipients to study the expression of ICAM-1, VCAM-1, specific apoptotic markers and the 6A3-5 gene expression, which is a new transcription factor upregulated by ischemia. Conclusions: The preliminary clinical and histological results of this pilot study seem to confirm our experimental investigations. Although the present results show that IGL-1 in kidney preservation is at least equivalent to UW solution with a prompter graft function and less DGF rate, a larger number of kidneys seem to be necessary to show IGL-1 superiority which has been reported in liver transplantation. A multicentric randomized study will show whether IGL-1 may be the solution of preference in multiorgan cadaveric storage.