Preservation of recipient plasma sphingosine-1-phosphate levels reduces transfusion-related acute lung injury.

Cold-stored (CS) platelets are once again being reintroduced for clinical use. Transfused CS platelets offer benefits over room temperature-stored (RTS) platelets such as increased hemostatic effects and prolongation of shelf-life. Despite these advantages little is known about their association with transfusion-related acute lung injury (TRALI). TRALI is associated with prolonged storage of RTS platelets and has a mortality of >15%. Determining the safety of CS platelets is important considering their proposed use in TRALI-vulnerable populations with inflammation such as surgical patients or patients with trauma. Donor platelet-derived ceramide causes TRALI, whereas donor platelet sphingosine-1-phosphate (S1P) is barrier protective. Females have higher plasma levels of S1P than males. Cold temperatures increase S1P levels in cells. Therefore, we hypothesized that female (donors or recipients) and/or CS platelets would decrease TRALI. To test this, we compared how male and female donor and recipient allogeneic platelet transfusions of CS (4°C) versus RTS (23°C) platelets stored for 5 days influence murine TRALI. Transfusion of CS platelets significantly reduced recipient lung tissue wet-to-dry ratios, bronchoalveolar lavage total protein, lung tissue myeloperoxidase enzyme activity, histological lung injury scores, and increased plasma sphingosine-1-phosphate (S1P) levels compared with RTS platelet transfusions. Female as opposed to male recipients had less TRALI and higher plasma S1P levels. Female donor mouse platelets had higher S1P levels than males. Mouse and human CS platelets had increased S1P levels compared with RTS platelets. Higher recipient plasma S1P levels appear protective considering females, and males receiving platelets from females or male CS platelets had less TRALI.NEW & NOTEWORTHY Transfusion-related acute lung injury (TRALI) though relatively rare represents a severe lung injury. The sphingolipid sphingosine-1-phosphate (S1P) regulates the severity of platelet-mediated TRALI. Female platelet transfusion recipient plasmas or stored platelets from female donors have higher S1P levels than males, which reduces TRALI. Cold storage of murine platelets preserves platelet-S1P, which reduces TRALI in platelet-transfused recipients.

Comparison of the effects of normothermic machine perfusion and cold storage preservation on porcine intestinal allograft regenerative potential and viability.

Intestinal transplantation (IT) is the final treatment option for intestinal failure. Static cold storage (CS) is the standard preservation method used for intestinal allografts. However, CS and subsequent transplantation induce ischemia-reperfusion injury (IRI). Severe IRI impairs epithelial barrier function, including loss of intestinal stem cells (ISC), critical to epithelial regeneration. Normothermic machine perfusion (NMP) preservation of kidney and liver allografts minimizes CS-associated IRI; however, it has not been used clinically for IT. We hypothesized that intestine NMP would induce less epithelial injury and better protect the intestine's regenerative ability when compared with CS. Full-length porcine jejunum and ileum were procured, stored at 4 °C, or perfused at 34 °C for 6 hours (T6), and transplanted. Histology was assessed following procurement (T0), T6, and 1 hour after reperfusion. Real-time quantitative reverse transcription polymerase chain reaction, immunofluorescence, and crypt culture measured ISC viability and proliferative potential. A greater number of NMP-preserved intestine recipients survived posttransplant, which correlated with significantly decreased tissue injury following 1-hour reperfusion in NMP compared with CS samples. Additionally, ISC gene expression, spheroid area, and cellular proliferation were significantly increased in NMP-T6 compared with CS-T6 intestine. NMP appears to reduce IRI and improve graft regeneration with improved ISC viability and proliferation.

Thyroid hormone protects human lung epithelial cells from cold preservation and warm reperfusion-induced injury.

BACKGROUND: Cellular stress associated with static-cold storage (SCS) and warm reperfusion of donor lungs can contribute to ischemia-reperfusion (IR) injury during transplantation. Adding cytoprotective agents to the preservation solution may be conducive to reducing graft deterioration and improving post-transplant outcomes. METHODS: SCS and warm reperfusion were simulated in human lung epithelial cells (BEAS-2B) by exposing cells to low potassium dextran glucose solution at 4 °C for different periods and then switching back to serum-containing culture medium at 37 °C. Transcriptomic analysis was used to explore potential cytoprotective agents. Based on its results, cell viability, caspase activity, cell morphology, mitochondrial function, and inflammatory gene expression were examined under simulated IR conditions with or without thyroid hormones (THs). RESULTS: After 18 h SCS followed by 2 h warm reperfusion, genes related to inflammation and cell death were upregulated, and genes related to protein synthesis and metabolism were downregulated in BEAS-2B cells, which closely mirrored gene profiles found in thyroid glands of mice with congenital hypothyroidism. The addition of THs (T3 or T4) to the preservation solution increases cell viability, inhibits activation of caspase 3, 8 and 9, preserves cell morphology, enhances mitochondrial membrane potential, reduces mitochondrial superoxide production, and suppresses inflammatory gene expression. CONCLUSION: Adding THs to lung preservation solutions may protect lung cells during SCS by promoting mitochondrial function, reducing apoptosis, and inhibiting pro-inflammatory pathways. Further in vivo testing is warranted to determine the potential clinical application of adding THs as therapeutics in lung preservation solutions.

Vesiculation in irradiated and cation-leaky-stored red blood cells.

BACKGROUND: Extracellular vesicles (EVs) are released by red blood cells (RBCs) throughout their life-span and also during hypothermic storage when they accumulate in the blood bag. We queried whether stored RBCs with increased cation permeability, either from donors with familial pseudohyperkalaemia (FP) or caused by irradiation, vesiculate more readily. STUDY DESIGN AND METHODS: Recent technical advances have revealed at least two sub-populations of MVs in RBC storage units: macrovesicles (2-6 µm) and microvesicles (1-2 µm). Using nanoparticle tracking analysis, imaging flow cytometry, and protein quantification methods, we measured and characterized vesicles released by RBCs from control and FP individuals at three different storage time-points (day 4, day 17, and day 29). The RBCs had either been stored untreated or irradiated on either day 1 or day 14 of storage. RESULTS: We found no difference in the number or size of vesicles released between cation-leaky FP RBCs and non-FP controls. Similarly, irradiated and non-irradiated RBCs showed very similar patterns of vesicle release to during cold-storage. The only significant difference in vesicle release was the increase in accumulated vesicles with length of storage time which has been reported previously. DISCUSSION: EVs in stored blood are potential contributors to adverse transfusion reactions. The number of vesicles released during 35-day hypothermic storage varies between donors and increases with storage duration. However, increased cation permeability and irradiation do not appear to affect vesicle formation during RBC cold-storage.

Two-day Static Cold Preservation of α1,3-Galactosyltransferase Knockout Kidney Grafts Before Simulated Xenotransplantation.

Transplantation remains the preferred treatment for end-stage kidney disease but is critically limited by the number of available organs. Xenografts from genetically modified pigs have become a promising solution to the loss of life while waiting for transplantation. However, the current clinical model for xenotransplantation will require off-site procurement, leading to a period of ischemia during transportation. As of today, there is limited understanding regarding the preservation of these organs, including the duration of viability, and the associated molecular changes. Thus, our aim was to evaluate the effects of static cold storage (SCS) on α1,3-galactosyltransferase knockout (GGTA1 KO) kidney. After SCS, viability was further assessed using acellular sub-normothermic ex vivo perfusion and simulated transplantation with human blood. Compared to baseline, tubular and glomerular interstitium was preserved after 2 days of SCS in both WT and GGTA1 KO kidneys. Bulk RNA-sequencing demonstrated that only eight genes were differentially expressed after SCS in GGTA1 KO kidneys. During sub-normothermic perfusion, kidney function, reflected by oxygen consumption, urine output, and lactate production was adequate in GGTA1 KO grafts. During a simulated transplant with human blood, macroscopic and histological assessment revealed minimal kidney injury. However, GGTA1 KO kidneys exhibited higher arterial resistance, increased lactate production, and reduced oxygen consumption during the simulated transplant. In summary, our study suggests that SCS is feasible for the preservation of porcine GGTA1 KO kidneys. However, alternative preservation methods should be evaluated for extended preservation of porcine grafts.

Cold Storage of Human Femoral Arteries for Twelve Months: Impact on Mechanical Properties.

OBJECTIVE: This biomechanical pre-clinical study aimed to assess the consequences on mechanical properties of long term cold storage (+2 to +8 °C) of arterial allografts. METHODS: Femoropopliteal arterial segments were collected from multiorgan donors and stored at +2 to +8 °C for twelve months in saline solution with added antibiotics. Mechanical characterisation was carried out using two different tests, with the aim of defining the physiological modulus and the maximum stress and strain borne by the sample before rupture. These characterisations were carried out after zero, six, and twelve months of storage for each sample (T0, T6, and T12, respectively). For comparison, the same tests were performed on cryopreserved femoropopliteal segments after thawing. RESULTS: Twelve refrigerated allografts (RAs), each divided into three segments, and 10 cryopreserved allografts (CAs) were characterised. The median (interquartile range [IQR]) Young's modulus was not statistically significantly different between the storage times for cold stored allografts: RAT0, 164 (150, 188) kPa; RAT6, 178 (141, 185) kPa; RAT12, 177 (149, 185) kPa. The median (IQR) Young's modulus of the CA group (153; 130, 170 kPa) showed no significant differences from the RA groups, irrespective of storage time. Furthermore, median (IQR) maximum stress and strain values were not significantly different between the different groups: for maximum stress: RAT0, 1.58 (1.08, 2.09) MPa; RAT6, 1.74 (1.55, 2.36) MPa; RAT12, 2.25 (1.87, 2.53) MPa; CA, 2.25 (1.77, 2.61) MPa; and for maximum strain: RAT0, 64% (50, 90); RAT6, 79% (63, 84); RAT12, 72% (65, 86); CA, 67% (50, 95). CONCLUSION: Cold storage for up to twelve months appears to have no impact on the mechanical characteristics of human arterial allografts. Therefore, this preservation method, which would greatly simplify routine care, seems feasible. Other indicators are being studied to verify the safety of this preservation process before considering its use in vivo.

The Predictive Value of Graft Viability and Bioenergetics Testing Towards the Outcome in Liver Transplantation.

Donor organ biomarkers with sufficient predictive value in liver transplantation (LT) are lacking. We herein evaluate liver viability and mitochondrial bioenergetics for their predictive capacity towards the outcome in LT. We enrolled 43 consecutive patients undergoing LT. Liver biopsy samples taken upon arrival after static cold storage were assessed by histology, real-time confocal imaging analysis (RTCA), and high-resolution respirometry (HRR) for mitochondrial respiration of tissue homogenates. Early allograft dysfunction (EAD) served as primary endpoint. HRR data were analysed with a focus on the efficacy of ATP production or P-L control efficiency, calculated as 1-L/P from the capacity of oxidative phosphorylation P and non-phosphorylating respiration L. Twenty-two recipients experienced EAD. Pre-transplant histology was not predictive of EAD. The mean RTCA score was significantly lower in the EAD cohort (-0.75 ± 2.27) compared to the IF cohort (0.70 ± 2.08; p = 0.01), indicating decreased cell viability. P-L control efficiency was predictive of EAD (0.76 ± 0.06 in IF vs. 0.70 ± 0.08 in EAD-livers; p = 0.02) and correlated with the RTCA score. Both RTCA and P-L control efficiency in biopsy samples taken during cold storage have predictive capacity towards the outcome in LT. Therefore, RTCA and HRR should be considered for risk stratification, viability assessment, and bioenergetic testing in liver transplantation.

Time Course of High-Energy Phosphate Depletion During Cold Storage of Human Heart Grafts Using the Celsior Solution.

The aim of this study was to provide insight into high-energy phosphate compound concentration dynamics under realistic clinical cold-storage conditions using the Celsior solution in seven heart grafts discarded from transplantation. The hearts of seven local donors (three males, four females, age 37 ± 17 years, height 175 ± 5 cm, weight 75 ± 9 kg) initially considered for transplantation and eventually discarded were submitted to a Magnetic Resonance Spectroscopy observation in a clinical Magnetic Resonance Imaging scanner over at least 9 h. The grafts remained in their sterile container at 4°C during the entire examination. Hence, Phosphocreatine (PCr), adenosine triphosphate (ATP), inorganic phosphate (Pi) and intracellular pH were recorded non-destructively at a 30-minute interval. With the ischemic time Ti, the concentration ratios decreased at PCr/ATP = 1.68-0.0028·Tis, Pi/ATP = 1.38 + 0.0029·Tis, and intracellular pH at 7.43-0.0012·Tis. ATP concentration remained stable for at least 9 h and did not decrease as long as phosphocreatine was detectable. Acidosis remained moderate. In addition to the standard parameters assessed at the time of retrieval, Magnetic Resonance Spectroscopy can provide an assesment of the metabolic status of heart grafts before transplantation. These results show how HEPC metabolites deplete during cold storage. Although many parameters determine graft quality during cold storage, the dynamics of HEPC and intracellular pH may be helpful in the development of strategies aiming at extending the ischemic time.

Involvement of energy and cell wall metabolisms in chilling tolerance improved by hydrogen sulfide in cold-stored tomato fruits.

KEY MESSAGE: Hydrogen sulfide improved cold resistance of tomato fruits by regulating energy metabolism and delaying cell wall degradation, thereby alleviating the damage of cold storage on fruits. Postharvest cold storage in tomato fruits extended shelf life but caused the appearance of chilling injury (CI), appeared by softness and spots on the surface of the fruits. These changes were linked closely with energy and cell wall metabolisms. Hydrogen sulfide (H2S), as the gaseous fresh-keeping regulator, was used in the present study to investigate the effects of H2S on energy and cell wall metabolisms in tomato fruits during cold storage. Fruits after harvest were fumigated with different concentrations (0, 0.5, 1, 1.5 mM) of sodium hydrosulfide (NaHS) solution as H2S honor for 24 h and stored at 4 °C for 25 days. The results showed that 1 and 1.5 mM NaHS solution fumigation promoted the accumulation of endogenous H2S, followed by the increase in L-cysteine desulfurase (LCD) and D-cysteine desulfurase (DCD) activities in fruits during cold storage. It was also found that 1 and 1.5 mM NaHS treatments improved H+-ATPase, Ca2+-ATPase, cytochrome C oxidase (CCO), and succinic dehydrogenase (SDH) activities. Moreover, the contents of cellulose and hemicellulose were increased by 1 and 1.5 mM NaHS, following down-regulated activities of cellulase (CL), pectin lyase (PL), α-mannosidase (α-man) and ß-Galactosidase (ß-Gal) and down-regulated expression of PL1, PL8, MAN4 and MAN7 genes. Thus, H2S alleviates CI led by cold storage in tomato fruits via regulating energy and cell wall metabolisms.

Oxygenated versus non-oxygenated flush out during deceased donor liver procurement: The first proof-of-concept study in humans.

BACKGROUND: Liver transplantation is used for treating end-stage liver disease, fulminant hepatitis, and oncological malignancies and organ shortage is a major limiting factor worldwide. The use of grafts based on extended donor criteria have become internationally accepted. Oxygenated machine perfusion technologies are the most recent advances in organ transplantation; however, it is only applied after a period of cold ischemia. Due to its high cost, we aimed to use a novel device, OxyFlush®, based on oxygenation of the preservation solution, applied during liver procurement targeting the maintenance of ATP during static cold storage (SCS). METHODS: Twenty patients were randomly assigned to the OxyFlush or control group based on a 1:1 ratio. In the OxyFlush group, the perfusion solution was oxygenated with OxyFlush® device while the control group received a non-oxygenated solution. Liver and the common bile duct (CBD) biopsies were obtained at three different time points. The first was at the beginning of the procedure, the second during organ preparation, and the third after total liver reperfusion. Biopsies were analyzed, and adenosine triphosphate (ATP) levels and histological scores of the liver parenchyma and CBD were assessed. Postoperative laboratory tests were performed. RESULTS: OxyFlush® was able to maintain ATP levels during SCS and improved the damage caused by the lack of oxygen in the CBD. However, OxyFlush® did not affect laboratory test results and histological findings of the parenchyma. CONCLUSION: We present a novel low-cost device that is feasible and could represent a valuable tool in organ preservation during SCS.

High-concentration bovine serum albumin enhances fertilization ability of cold-stored rat sperm.

Cold transport of the cauda epididymides is a useful technique for shipping laboratory rat sperm. Cold transport of rat sperm avoids potential risks of microbiological infection, animal escape or death, and animal welfare issues. Previously, we reported that a cold-storage solution containing dimethyl sulfoxide and quercetin maintained the fertility of cold-stored rat sperm. However, cold-stored rat sperm exhibited a decreased fertilization rate after 24-h storage. To recover the fertility of cold-stored sperm, we focused on the effects of bovine serum albumin (BSA), a cholesterol acceptor that induces sperm capacitation. We sought to determine the optimal concentration of BSA in fertilization medium based on the fertility of cold-stored rat sperm. High concentrations of BSA (40 mg/ml) enhanced the fertilization rate of cold-stored rat sperm and maintained sperm fertility for 144 h. Embryos derived from cold-stored and BSA-treated sperm normally developed into pups after embryo transfer. In summary, high BSA concentrations enhanced the fertility of cold-stored rat sperm and prolonged the storage period to 144 h, thereby expanding the transportable region for genetically engineered rats.

The platelet storage lesion, what are we working for?

BACKGROUND: Platelet concentrate (PC) transfusions are crucial in prevention and treatment of bleeding in infection, surgery, leukemia, and thrombocytopenia patients. Although the technology for platelet preparation and storage has evolved over the decades, there are still challenges in the demand for platelets in blood banks because the platelet shelf life is limited to 5 days due to bacterial contamination and platelet storage lesions (PSLs) at 20-24°C under constant horizontal agitation. In addition, the relations between some adverse effects of platelet transfusions and PSLs have also been considered. Therefore, understanding the mechanisms of PSLs is conducive to obtaining high quality platelets and facilitating safe and effective platelet transfusions. OBJECTIVE: This review summarizes developments in mechanistic research of PSLs and their relationship with clinical practice, providing insights for future research. METHODS: Authors conducted a search on PubMed and Web of Science using the professional terms "PSL" and "platelet transfusion." The obtained literature was then roughly categorized based on their research content. Similar studies were grouped into the same sections, and further searches were conducted based on the keywords of each section. RESULTS: Different studies have explored PSLs from various perspectives, including changes in platelet morphology, surface molecules, biological response modifiers (BMRs), metabolism, and proteins and RNA, in an attempt to monitor PSLs and identify intervention targets that could alleviate PSLs. Moreover, novel platelet storage conditions, including platelet additive solutions (PAS) and reconsidered cold storage methods, are explored. There are two approaches to obtaining high-quality platelets. One approach simulates the in vivo environment to maintain platelet activity, while the other keeps platelets at a low activity level in vitro under low temperatures. CONCLUSION: Understanding PSLs helps us identify good intervention targets and assess the therapeutic effects of different PSLs stages for different patients.

Fungicide-Resistant Botrytis in Forest Nurseries May Impact Disease Control in Norway Spruce.

Gray mold, caused by Botrytis spp., is a serious problem in Norway spruce seedling production in forest nurseries. From 2013 to 2019, 125 isolates of Botrytis were obtained from eight forest nurseries in Norway: 53 from Norway spruce seedlings, 16 from indoor air, 52 from indoor surfaces, and four from weeds growing close to seedlings. The majority of isolates were identified as B. cinerea, and over 60% of these were characterized as Botrytis group S. B. pseudocinerea isolates were obtained along with isolates with DNA sequence similarities to B. prunorum. Fungicide resistance was assessed with a mycelial growth assay, and resistance was found for the following: boscalid (8.8%), fenhexamid (33.6%), fludioxonil (17.6%), pyraclostrobin (36.0%), pyrimethanil (13.6%), and thiophanate-methyl (50.4%). Many isolates (38.4%) were resistant to two to six different fungicides. A selection of isolates was analyzed for the presence of known resistance-conferring mutations in the cytb, erg27, mrr1, sdhB, and tubA genes, and mutations leading to G143A, F412S, ΔL497, H272R, and E198A/F200Y were detected, respectively. Detection of fungicide resistance in Botrytis from Norway spruce and forest nursery facilities reinforces the necessity of employing resistance management strategies to improve control and delay development of fungicide resistance in the gray mold pathogens.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

In vitro Hemostatic Functions of Cold-Stored Platelets.

Background: Transfusion of platelets is a life-saving medical strategy used worldwide to treat patients with thrombocytopenia as well as platelet function disorders. Summary: Until the end of 1960s, platelets were stored in the cold because of their superior hemostatic functionality. Cold storage of platelets was then abandoned due to better posttransfusion recovery and survival of room temperature (RT)-stored platelets, demonstrated by radioactive labeling studies. Based on these findings, RT became the standard condition to store platelets for clinical applications. Evidence shows that RT storage increases the risk of septic transfusion reactions associated with bacterial contamination. Therefore, the storage time is currently limited to 4-7 days, according to the national guidelines, causing a constant challenge to cover the clinical request. Despite the enormous efforts made to optimize storage conditions of platelets, the quality and efficacy of platelets still decrease during the short storage time at RT. In this context, during the last years, cold storage has seen a renaissance due to the better hemostatic functionality, reduced risk of bacterial contamination, and potentially longer storage time. Key Messages: In this review, we will focus on the impact of cold storage on the in vitro platelet functions as promising alternative storage temperature for future medical applications.

Identification of the Key Gene DfCCoAOMT1 through Comparative Analysis of Lignification in Dendrocalamus farinosus XK4 and ZPX Bamboo Shoots during Cold Storage.

Dendrocalamus farinosus bamboo shoots, a species with rich nutritional value, are important in Southwest China. Lignin is an important factor affecting the postharvest flavor quality of bamboo shoots; however, the underlying mechanism of lignin deposition in D. farinosus bamboo shoots during cold storage is still not fully understood. In this study, the mutant D. farinosus XK4 with low lignin content at 3.11% and the cultivated variety ZPX at 4.47% were used as experimental materials. The lignin content of D. farinosus XK4 and ZPX, as well as the gene expression differences between them, were compared and analyzed during cold storage using transcriptomic and physiological methods. Our analysis revealed several key genes and found that D. farinosus CCoAOMT1 plays a key role in the regulatory network of bamboo shoots during cold storage. Tobacco heterologous transformation experiments demonstrated that overexpression of DfCCoAOMT1 significantly increases lignin content. This study provides a novel foundation for future research aimed at improving the postharvest quality and flavor of D. farinosus bamboo shoots through targeted genetic manipulation during cold storage.

Enhancing Heart Transplantation: Utilizing Gas-Loaded Nanocarriers to Mitigate Cold/Hypoxia Stress.

Gas-loaded nanocarriers (G-LN) show promise in improving heart transplantation (HTx) outcomes. Given their success in reducing cell death during normothermic hypoxia/reoxygenation (H/R) in vitro, we tested their integration into cardioplegic solutions and static cold storage (SCS) during simulated HTx. Wistar rat hearts underwent four hours of SCS with four G-LN variants: O2- or N2-cyclic-nigerosyl-nigerose-nanomonomers (CNN), and O2- or N2-cyclic-nigerosyl-nigerose-nanosponges (CNN-NS). We monitored physiological-hemodynamic parameters and molecular markers during reperfusion to assess cell damage/protection. Hearts treated with nanomonomers (N2-CNN or O2-CNN) showed improvements in left ventricular developed pressure (LVDP) and a trend towards faster recovery of the rate pressure product (RPP) compared to controls. However, nanosponges (N2-CNN-NS or O2-CNN-NS) did not show similar improvements. None of the groups exhibited an increase in diastolic left ventricular pressure (contracture index) during reperfusion. Redox markers and apoptosis/autophagy pathways indicated an increase in Beclin 1 for O2-CNN and in p22phox for N2-CNN, suggesting alterations in autophagy and the redox environment during late reperfusion, which might explain the gradual decline in heart performance. The study highlights the potential of nanomonomers to improve early cardiac performance and mitigate cold/H/R-induced stunning in HTx. These early improvements suggest a promising avenue for increasing HTx success. Nevertheless, further research and optimization are needed before clinical application.

Evaluating the Effects of Kidney Preservation at 10 °C with Hemopure and Sodium Thiosulfate in a Rat Model of Syngeneic Orthotopic Kidney Transplantation.

Kidney transplantation is preferred for end-stage renal disease. The current gold standard for kidney preservation is static cold storage (SCS) at 4 °C. However, SCS contributes to renal graft damage through ischemia-reperfusion injury (IRI). We previously reported renal graft protection after SCS with a hydrogen sulfide donor, sodium thiosulfate (STS), at 4 °C. Therefore, this study aims to investigate whether SCS at 10 °C with STS and Hemopure (blood substitute), will provide similar protection. Using in vitro model of IRI, we subjected rat renal proximal tubular epithelial cells to hypoxia-reoxygenation for 24 h at 10 °C with or without STS and measured cell viability. In vivo, we preserved 36 donor kidneys of Lewis rats for 24 h in a preservation solution at 10 °C supplemented with STS, Hemopure, or both followed by transplantation. Tissue damage and recipient graft function parameters, including serum creatinine, blood urea nitrogen, urine osmolality, and glomerular filtration rate (GFR), were evaluated. STS-treated proximal tubular epithelial cells exhibited enhanced viability at 10 °C compared with untreated control cells (p < 0.05). Also, STS and Hemopure improved renal graft function compared with control grafts (p < 0.05) in the early time period after the transplant, but long-term function did not reach significance. Overall, renal graft preservation at 10 °C with STS and Hemopure supplementation has the potential to enhance graft function and reduce kidney damage, suggesting a novel approach to reducing IRI and post-transplant complications.

Mitigating Cold Ischemic Injury: HTK, UW and IGL-2 Solution's Role in Enhancing Antioxidant Defence and Reducing Inflammation in Steatotic Livers.

Liver transplantation remains the only definitive treatment for end-stage liver diseases. However, the increasing prevalence of fatty liver disease among potential donors exacerbates the shortage of suitable organs. This study evaluates the efficacy of the preservation solution Institut Georges Lopez-2 (IGL-2) compared to Histidine-Tryptophan-Ketoglutarate (HTK) and University of Wisconsin (UW) preservation solutions in mitigating ischemia-reperfusion injury (IRI) in steatotic livers. Using Zucker Obese rat livers, we assessed the impact of 24-h static cold storage (SCS) with each solution on transaminase release, glutathione redox balance, antioxidant enzyme activity, lipoperoxidation, and inflammation markers. IGL-2 and UW solutions demonstrated reduced transaminase and lactate levels compared to HTK, indicating better preservation of liver integrity. IGL-2 maintained a higher reduced glutathione/oxidized glutathione (GSH/GSSG) ratio, suggesting more effective management of oxidative stress. Antioxidant enzyme activities catalase, superoxide dismutase, and glutathione peroxidase (CAT, SOD, GPX) were higher in IGL-2 preserved livers, contributing to decreased oxidative damage. Lipid peroxidation markers and inflammatory markers were lower in IGL-2 than in HTK, indicating reduced oxidative stress and inflammation. Additionally, improved mitochondrial function was observed in the IGL-2 group, correlating with reduced reactive oxygen species (ROS) production and lipid peroxidation. These findings suggest that IGL-2 offers superior preservation of liver viability, reduces oxidative stress, and minimizes inflammation compared to HTK and UW solutions. By maintaining a higher ratio of reduced glutathione and antioxidant enzyme activity, IGL-2 effectively mitigates the harmful effects of ischemia-reperfusion injury. The reduced lipid peroxidation and inflammation in the IGL-2 group further underscore its potential in improving liver transplant outcomes. These results highlight the importance of optimizing preservation solutions to enhance the viability and functionality of donor organs, potentially expanding the donor pool and improving the success rates of liver transplantation. Future research should focus on refining preservation techniques and exploring additional protective agents to further improve organ preservation and transplant outcomes.

Normal Proteasome Function Is Needed to Prevent Kidney Graft Injury during Cold Storage Followed by Transplantation.

Kidney transplantation is the preferred treatment for end-stage kidney disease (ESKD). However, there is a shortage of transplantable kidneys, and donor organs can be damaged by necessary cold storage (CS). Although CS improves the viability of kidneys from deceased donors, prolonged CS negatively affects transplantation outcomes. Previously, we reported that renal proteasome function decreased after rat kidneys underwent CS followed by transplantation (CS + Tx). Here, we investigated the mechanism underlying proteasome dysfunction and the role of the proteasome in kidney graft outcome using a rat model of CS + Tx. We found that the key proteasome subunits ß5, α3, and Rpt6 are modified, and proteasome assembly is impaired. Specifically, we detected the modification and aggregation of Rpt6 after CS + Tx, and Rpt6 modification was reversed when renal extracts were treated with protein phosphatases. CS + Tx kidneys also displayed increased levels of nitrotyrosine, an indicator of peroxynitrite (a reactive oxygen species, ROS), compared to sham. Because the Rpt6 subunit appeared to aggregate, we investigated the effect of CS + Tx-mediated ROS (peroxynitrite) generation on renal proteasome assembly and function. We treated NRK cells with exogenous peroxynitrite and evaluated PAC1 (proteasome assembly chaperone), Rpt6, and ß5. Peroxynitrite induced a dose-dependent decrease in PAC1 and ß5, but Rpt6 was not affected (protein level or modification). Finally, serum creatinine increased when we inhibited the proteasome in transplanted donor rat kidneys (without CS), recapitulating the effects of CS + Tx. These findings underscore the effects of CS + Tx on renal proteasome subunit dysregulation and also highlight the significance of proteasome activity in maintaining graft function following CS + Tx.

Effect of Sodium Thiosulfate Pre-Treatment on Renal Ischemia-Reperfusion Injury in Kidney Transplantation.

We recently reported in a rat model of kidney transplantation that the addition of sodium thiosulfate (STS) to organ preservation solution improved renal graft quality and prolonged recipient survival. The present study investigates whether STS pre-treatment would produce a similar effect. In vitro, rat kidney epithelial cells were treated with 150 µM STS before and/or during exposure to hypoxia followed by reoxygenation. In vivo, donor rats were treated with PBS or 2.4 mg/kg STS 30 min before donor kidneys were procured and stored in UW or UW+150 µM STS solution at 4 °C for 24 h. Renal grafts were then transplanted into bilaterally nephrectomised recipient rats which were then sacrificed on post-operative day 3. STS pre-treatment significantly reduced cell death compared to untreated and other treated cells in vitro (p < 0.05), which corresponded with our in vivo result (p < 0.05). However, no significant differences were observed in other parameters of tissue injury. Our results suggest that STS pre-treatment may improve renal graft function after transplantation.