Kidney transplantation is critically limited by the shortage of available donor organs, exacerbated by the discarding of viable organs due to insufficient allocation time. Here, we demonstrate the application of nature-inspired partial freezing for long-term storage of kidney grafts. We show that, after 10 days of storage, partially frozen swine and human kidneys retain improved function compared to those preserved using traditional static cold storage, as assessed via simulated transplantation.

Females improved resilience to ischemia-reperfusion injury has been reported in animal models, a protection largely attributed to sexual hormones. Clinically, the impact of sex on short- and long-term outcomes after kidney transplantation is unclear. In this retrospective cohort study using the United Network for Organ Sharing database, we analyzed paired donor kidneys transplanted to 2 opposite sex adult recipients (N = 39 197 males and N = 39 197 females), thereby controlling for all donor-related covariates. The primary endpoint was delayed graft function. Secondary outcomes included 1-year graft and patient survival and estimated glomerular filtration rate at 6 months and 1 year. We adjusted the results for the following covariates: years receiving dialysis, human leukocyte antigen mismatch, calculated panel reactive antibodies, and cold ischemic time. Consistent with preclinical data, female donor and recipient sex was associated with reduced delayed graft function, especially in the context of donation after cardiocirculatory death (F-to-M: odds ratio [OR] = 0.73, M-to-F: OR = 0.59, F-to-F: OR = 0.39, reference: M-to-M; P < .0001 for all comparisons). Female recipients had improved 1-year graft and patient survival, while male donor sex was associated with improved 1-year estimated glomerular filtration rate, independently of donor size. These findings highlight the role of sex as a clinically relevant biological variable influencing transplant outcomes.

Aging is accompanied by a decline in neovascularization potential and increased susceptibility to ischemic injury. Here, we confirm the age-related impaired neovascularization following ischemic leg injury and impaired angiogenesis. The age-related deficits in angiogenesis arose primarily from diminished EC proliferation capacity, but not migration or VEGF sensitivity. Aged EC harvested from the mouse skeletal muscle displayed a pro-angiogenic gene expression phenotype, along with considerable changes in metabolic genes. Metabolomics analysis and 13C glucose tracing revealed impaired ATP production and blockade in glycolysis and TCA cycle in late passage HUVECs, which occurred at nicotinamide adenine dinucleotide (NAD')-dependent steps, along with NAD+ depletion. Supplementation with nicotinamide mononucleotide (NMN), a precursor of NAD', enhances late-passage EC proliferation and sprouting angiogenesis from aged mice aortas. Taken together, our study illustrates the importance of NAD+-dependent metabolism in the maintenance of EC proliferation capacity with age, and the therapeutic potential of NAD precursors.

Organ transplantation is the only definitive cure for end-stage organ failure. The primary obstacle is the scarcity of viable donor organs, resulting in many patients dying on the transplant waitlist. The last decade has brought a range of transformative technologies to address this shortage: machine perfusion has risen as an option to greatly increase the human donor organ utilization, while xenotransplantation further promises to fundamentally alter the calculus in supply constraints. Given this increasing availability, the logistics of allocation rises as the next key technological barrier. Cryopreservation strategies aim to increase the viable storage time for organs and thereby enable the creation of a cold-supply chain for clinical transplantation. This technology has the potential to overcome limitations related to preservation and transportation, histocompatibility matching, complex organ allocation and wastage, and in tandem with xenotransplantation, provide an unlimited off-the-shelf supply of organs. This review aims to cover the latest research updates in the use of cryopreservation, how it compares to current clinical preservation strategies, and discuss how its implementation into the world of transplantation may require new logistical, ethical and regulatory frameworks.

INTRODUCTION:Metabolic alterations are recognized as key features of kidney injury, but their causal role in kidney repair remains debatable. Here, we investigate the role of phosphoenolpyruvate carboxykinase 1 (PCK1), an enzyme involved in gluconeogenesis and cataplerosis (removal of tricarboxylic acid (TCA) cycle intermediates from the mitochondrial matrix) in kidney disease progression. METHODS:We used mice with kidney tubular cell-specific deletion or overexpression of the PCK1 enzyme, and different models of kidney injury such as ischemia-reperfusion injury or cis-platin-induced nephropathy. Furthermore, we measured metabolites in kidney biopsy tissue from patients with stage 3b/4 chronic kidney disease (CKD). RESULTS:Using flux analysis, we confirm that cataplerosis and the TCA cycle are blocked by PCK1 deficiency. This results in injured mitochondria leading to inflammation, tubular injury and impaired tubular cell repair. Inversely, maintaining PCK1 function in different models of kidney injury preserves kidney structure, improves TCA cycle metabolite clearance and increase ATP production. In kidney biopsies from different patient cohorts, we confirm the correlation between PCK1 loss, mitochondrial injury and a failed tubular cell repair phenotype. Furthermore, in CKD, accumulation of TCA cycle metabolites is consistent with disrupted cataplerosis. CONCLUSIONS:Overall, we demonstrate that PCK1 loss in kidney tubular cells leads to decreased respiration and the accumulation of TCA cycle metabolites. Maintenance of cataplerosis is an important factor of tubular physiology and repair, with PCK1 serving as a causal and potential therapeutic target in this process. PCK1 restoration enhances mitochondrial health, limiting progression to inflammation and fibrosis.

Static cold storage of donor livers at 4°C incompletely arrests metabolism, ultimately leading to decreases in ATP levels, oxidative stress, cell death, and organ failure. Hydrogen Sulfide (H2S) is an endogenously produced gas, previously demonstrated to reduce oxidative stress, reduce ATP depletion, and protect from ischemia and reperfusion injury. H2S is difficult to administer due to its rapid release curve, resulting in cellular death at high concentrations. AP39, a mitochondrially targeted, slow-release H2S donor, has been shown to reduce ischemia-reperfusion injury in hearts and kidneys. Thus, we investigated whether the addition of AP39 during 3-day static cold storage can improve liver graft viability. At the end of storage, livers underwent six hours of acellular normothermic machine perfusion, a model of transplantation. During simulated transplantation, livers stored with AP39 showed reduced resistance, reduced cellular damage (ALT and AST), and reduced apoptosis. Additionally, bile production and glucose, as well as energy charge were improved by the addition of AP39. These results indicate that AP39 supplementation improves liver viability during static cold storage.

Background. Hydrogen sulfide (H2S) produced endogenously by the CTH gene-encoded cystathionine gamma-lyase protects from renal ischemia–reperfusion injury in preclinical models. Here, we hypothesized that CTH gene polymorphisms (single nucleotide polymorphism [SNP]) and recipient H2S serum levels influence kidney graft outcomes after transplantation. Methods. We included all consecutive recipients of a first kidney transplant in the Swiss Transplant Cohort Study and with available genotyping. In addition, 192 deceased-donor kidney transplant recipients were randomly selected to measure baseline serum H2S levels. The primary endpoint was graft loss during follow-up. Results. CTH SNPs were identified in up to 50% of the patients. During median follow-up (6.4 y, interquartile range: 3.9–9.8), graft loss was observed in 247 (9.8%) of 2518 patients. The incidence of graft loss was associated with the presence or absence of CTH SNPs. Specifically, rs672203 and rs10458561, increased the risk of graft loss (hazard ratio [HR]: 1.36, 95% confidence interval [CI]: 1.04-1.78, P = 0.02; and HR: 1.29, 95% CI: 1.0-1.66, P = 0.05; respectively), whereas rs113285275 was protective (HR: 0.78, 95% CI: 0.6-1.01, P = 0.05). Interestingly, rs672203 was associated with an increased risk of acute rejection (P = 0.05), whereas rs113285275 was associated with a lower risk of acute rejection (P = 0.01). Finally, in patients with delayed graft function, serum H2S levels correlated with lower graft dysfunction (defined as estimated glomerular filtration rate <30 mL/min/1.73 m2) (P = 0.05). Conclusions. Graft outcome after kidney transplantation was associated with CTH genotype and, to some extent, H2S serum levels. Further research is needed to define the underlying protective mechanisms.

One-week protein restriction (PR) limits ischemia-reperfusion (IR) damages and improves metabolic fitness. Similarly, longer-term calory restriction results in increased lifespan, partly via reduced insulin-like growth factor (IGF)-1. However, the influence of short-term PR on IGF-1 and its impact on IR are unknown. PR was achieved in mice via one-week carbohydrate loading and/or through a low-protein diet. PR decreased IGF-1 circulating levels as well as renal and hepatic expression. Upon renal IR, serum IGF-1 positively correlated with renal dysfunction and tissular damages, independently of sex and age. Exogenous IGF-1 administration abrogated PR benefits during IR, while IGF-1 receptor inhibition with linsitinib was protective. IGF-1 was associated with a reduction in forkhead box O (FoxO), and AMP-activated protein kinase (AMPK) signaling pathways previously demonstrated to improve IR resilience in various organs. These data support dietary or pharmacological reduction of IGF-1 signaling to mitigate IR injury prior to solid organ transplantation and beyond.

The vascular system experiences an age-associated decline in tissue perfusion and response to ischemic diseases. The factors driving this age-associated neovascularization decline remain unclear. While old endothelial cells (ECs) adopt a pro-angiogenic gene expression profile, we observed a stark reduction in the proliferative capacity of old ECs, while migratory capabilities remain intact. This is paralleled by a drastic decline in glycolytic capacity and ATP production, which likely act as limiting factors to neovascularization. These findings may provide new strategies to restore EC function in aging, thereby improving organ resilience and extending health- and lifespan.### Competing Interest StatementThe authors have declared no competing interest.

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.

Dietary restriction promotes resistance to surgical stress in multiple organisms. Counterintuitively, current medical protocols recommend short-term carbohydrate-rich drinks (carbohydrate loading) prior to surgery, part of a multimodal perioperative care pathway designed to enhance surgical recovery. Despite widespread clinical use, preclinical and mechanistic studies on carbohydrate loading in surgical contexts are lacking. Here we demonstrate in ad libitum -fed mice that liquid carbohydrate loading for one week drives reductions in solid food intake, while nearly doubling total caloric intake. Similarly, in humans, simple carbohydrate intake is inversely correlated with dietary protein intake. Carbohydrate loading-induced protein dilution increases expression of hepatic fibroblast growth factor 21 (FGF21) independent of caloric intake, resulting in protection in two models of surgical stress: renal and hepatic ischemia-reperfusion injury. The protection is consistent across male, female, and aged mice. In vivo, amino acid add-back or genetic FGF21 deletion blocks carbohydrate loading-mediated protection from ischemia-reperfusion injury. Finally, carbohydrate loading induction of FGF21 is associated with the induction of the canonical integrated stress response (ATF3/4, NF-kB), and oxidative metabolism (PPARγ). Together, these data support carbohydrate loading drinks prior to surgery and reveal an essential role of protein dilution via FGF21.

Background. In rodents, hydrogen sulfide (H2S) reduces ischemia-reperfusion injury and improves renal graft function after transplantation. Here, we hypothesized that the benefits of H2S are conserved in pigs, a more clinically relevant model. Methods. Adult porcine kidneys retrieved immediately or after 60 min of warm ischemia (WI) were exposed to 100 µM sodium hydrosulfide (NaHS) (1) during the hypothermic ex vivo perfusion only, (2) during WI only, and (3) during both WI and ex vivo perfusion. Kidney perfusion was evaluated with dynamic contrast-enhanced MRI. MRI spectroscopy was further employed to assess energy metabolites including ATP. Renal biopsies were collected at various time points for histopathological analysis. Results. Perfusion for 4 h pig kidneys with Belzer MPS UW + NaHS resulted in similar renal perfusion and ATP levels than perfusion with UW alone. Similarly, no difference was observed when NaHS was administered in the renal artery before ischemia. After autotransplantation, no improvement in histologic lesions or cortical/medullary kidney perfusion was observed upon H2S administration. In addition, AMP and ATP levels were identical in both groups. Conclusions. In conclusion, treatment of porcine kidney grafts using NaHS did not result in a significant reduction of ischemia-reperfusion injury or improvement of kidney metabolism. Future studies will need to define the benefits of H2S in human, possibly using other molecules as H2S donors.

Background. The ideal preservation temperature for donation after circulatory death kidney grafts is unknown. We investigated whether subnormothermic (22 °C) ex vivo kidney machine perfusion could improve kidney metabolism and reduce ischemia-reperfusion injury. Methods. To mimic donation after circulatory death procurement, kidneys from 45-kg pigs underwent 60 min of warm ischemia. Kidneys were then perfused ex vivo for 4 h with Belzer machine perfusion solution UW at 22 °C or at 4 °C before transplantation. Magnetic resonance spectroscopic imaging coupled with LCModel fitting was used to assess energy metabolites. Kidney perfusion was evaluated with dynamic-contrast enhanced MRI. Renal biopsies were collected at various time points for histopathologic analysis. Results. Total adenosine triphosphate content was 4 times higher during ex vivo perfusion at 22 °C than at 4 °C perfusion. At 22 °C, adenosine triphosphate levels increased during the first hours of perfusion but declined afterward. Similarly, phosphomonoesters, containing adenosine monophosphate, were increased at 22 °C and then slowly consumed over time. Compared with 4 °C, ex vivo perfusion at 22 °C improved cortical and medullary perfusion. Finally, kidney perfusion at 22 °C reduced histological lesions after transplantation (injury score: 22 °C: 10.5 ± 3.5; 4 °C: 18 ± 2.25 over 30). Conclusions. Ex vivo kidney perfusion at 22°C improved graft metabolism and protected from ischemia-reperfusion injuries upon transplantation. Future clinical studies will need to define the benefits of subnormothermic perfusion in improving kidney graft function and patient’s survival.