Preserving and rejuvenating old organs for transplantation: Novel treatments including the potential of senolytics

Abstract

Purpose of review

Older donors have the potential to close the gap between demand and supply in solid organs transplantation. Utilizing older organs, at the same time, has been associated with worse short- and long-term outcomes. Here, we introduce potential mechanisms on how treatments during machine perfusion (MP) may safely improve the utilization of older organs.

Recent findings

Consequences of ischemia reperfusion injury (IRI), a process of acute, sterile inflammation leading to organ injury are more prominent in older organs. Of relevance, organ age and IRI seem to act synergistically, leading to an increase of damage associated molecular patterns that trigger innate and adaptive immune responses. While cold storage has traditionally been considered the standard of care in organ preservation, accumulating data support that both hypothermic and normothermic MP improve organ quality, particularly in older organs. Furthermore, MP provides the opportunity to assess the quality of organs while adding therapeutic agents. Experimental data have already demonstrated the potential of applying treatments during MP. New experimental show that the depletion of senescent cells that accumulate in old organs improves organ quality and transplant outcomes.

Summary

As the importance of expanding the donor pool is increasing, MP and novel treatments bear the potential to assess and regenerate older organs, narrowing the gap between demand and supply.

Introduction

In parallel to changing demographics, donor age has increased for all solid organ transplants over the past decade. (optn.transplant.hrsa.gov/data/view-data-reports/national-data, accessed on 6.30.2022). Donor age, at the same time, significantly increases the risk of adverse outcomes linked to an advanced organ damage in addition to an organ-age related augmented immunogenicity (1-3). As demand still faces limited supply, optimal utilization of old organs seems critical to reduce waiting times, morbidity, and mortality rates of patients awaiting organ transplantation (2, 3).

The consequences of ischemia-reperfusion injury (IRI) have been proven to advance synergistically with rising donor age and prolonged cold ischemic time, clinically manifested by more frequent rates of delayed graft function (DGF) (4-7). Moreover, DGF appears to be associated with an elevated risk of acute rejections following transplantation, impairing both short- and long-term outcomes (8, 9).

In recent years, machine perfusion (MP) has gained clinical relevance, yielding superior short-term outcomes with lower DGF rates compared to static cold storage (CS). MP not only contributes to improved preservation of the graft but may also facilitate assessment and potentially treatment during the preservation period (10*, 11).

Here, we present both experimental and clinical data on age-specific aspects of organ damage and assess potential treatment methods within MP when using older organs for transplantation.

Donor age effects on transplant outcomes

Globally changing demographics with a rising older population have impacted the availability of organs for transplantation. As older organs bear the potential of closing the gap between demand and supply, optimal utilization of those organs is urgently needed to expand the donor pool (12-14). At the same time, donor age has been associated with increased rejection rates and more advanced organ injuries subsequent to IRI, all leading to compromised transplant outcomes (2, 3, 15).

Clinically, donor age seems to negatively affect outcomes across all solid organ transplantations. In lung transplantation, grafts from donors age > 65 years have shown increased 1-year graft failure rates. Interestingly, inferior outcomes of lungs procured from donors aged 56-65 were only observed when transplanted into severely ill recipients, emphasizing on the importance of donor recipient matching (16). An extensive analysis of the United Network for Organ Sharing (UNOS) registry demonstrated poorer survival rates with incrementally increasing older donor organs (>40 years) (17). Comparably seen in liver transplantation, an analysis of almost 35.000 transplants listed with the European Liver Transplant Registry demonstrated progressively worsening survival outcomes at both 3- and 12-months with increasing donor age (18). In renal transplantation, donor age and diabetes as the cause of renal failure have been identified as the main drivers of recipient death-censored survival in an analysis of > 50.000 deceased kidney transplantations (19, 20). Delineating the relevance and consequences of recipient and donor age among > than 300.000 renal transplant recipients listed in the UNOS data base, older kidneys demonstrated to have higher rates of acute rejections within the first post-transplant year. This finding is in line with our own observations that older organs have an augmented immunogenicity (2, 3). Indeed, recipients of organs from donors 60-69 years experienced a 24.5% rejection rate which was significantly higher compared to rates of younger donors (3).

Accordingly supported by experimental data, older organs appear to be more immunogenic due to an augmented T-cell alloreactivity, cytokine production, and elevated numbers of peripheral T- and B-cells(21).

Additional experimental data from our group have recently delineated the detrimental role of old dendritic cells (DCs) in augmenting alloimmune responses (22). In contrast young DCs have been able to enhance allograft survival while depleting intra-graft DCs prolonged allograft survival in old mice (23, 24). Mechanistically, we found that old organs contained increased numbers of senescent cells serving as a source of cell-free-mt-DNA (cf-mt-DNA). Increased cf-mt-DNA levels, in turn, promote DC-mediated amplification of Th1/Th17 alloimmune responses (22, 24). Those data have provided a rationale seeking ways to deplete senescent cells in old organs prior to transplantation to improve organ quality and transplant outcomes.

Aging increases the susceptibility of grafts to ischemia-reperfusion injury

IRI represents a process of acute, sterile inflammation occurring with the restoration of blood flow after an initial phase of ischemia (25). This multi-factorial process leads to a morphological changes involving broad molecular and cellular mechanisms (26). As a result of a complex interaction of different channels and transporters, calcium overload has been proposed to be an initial step in the pathogenesis of the injury (27, 28). Moreover, both ischemia itself and reperfusion lead to an overload of Reactive Oxygen Species (ROS) (29). Mechanistically, ROS disturb cellular functions and signaling pathways in addition to augmenting innate immune responses (30). Both, calcium- and ROS-overload initiate the formation of the mitochondrial permeability transition pore (mPTP) (31). This process leads to structural damage through mitochondrial swelling, allowing the influx of hydrogen ions while uncoupling the electron transport chain and accelerating ROS release upon reperfusion (32, 33). These changes also trigger both apoptosis and necrosis causing a further release of ROS, proinflammatory cytokines and damage-associated molecular patterns (34). Finally, disturbed cell and tissue integrity triggers an inflammatory cascade activating players of both, the innate and the adaptive immune system (35).

As an inevitable consequence of solid organ transplantation, IRI affects both short- and long-term outcomes (19, 36-39). Although effects on clinical outcomes have not been entirely delineated, there is strong experimental evidence that aging enhances the susceptibility of IRI in clinical transplantation (40, 41). Despite the minimal morphologic changes with age, older livers have significantly reduced intracellular energy content after IRI linked to a reduced capacity of mitochondrial ATP production (42). Recent investigations have been able to link the augmented IRI-susceptibility of older livers to a depletion of both sirtuin-1 and mitofusin-2 accompanied with defective autophagy and mPTP onset (43). In addition, an age-dependent mitophagy dysfunction may be at play. Mechanistically, defective mitochondria are removed via autophagy (=mitophagy) with the critical involvement of parkin, an E3 ubiquitin ligase, priming impaired mitochondria for removal. Immunohistochemistry analysis of DCD-graft biopsies performed 2-hours after revascularization revealed a low parkin expression with increasing donor age, a process linked to an augmented IRI-sensitivity (44). Of additional interest, pretreating old rats with pooled young plasma restored hepatic autophagic activity and reduced age-dependent liver IRI (45).

Comparable data are available for older kidneys showing an ineffective autophagy following IRI (46). Additionally, changes in blood urea nitrogen and creatinine appear more severe with aging including mitochondrial morphology, tubular epithelial loss and hemorrhage being significantly more pronounced in older kidneys (47, 48). Recent experimental data have shown that organ age and IRI act synergistically, leading to an increased DAMP release, triggering innate immune activation. Cf-mt-DNA levels acting as a DAMP were 15x higher in old mice after IRI compared to young mice (22). Of clinical relevance, several studies have shown increased levels of urinary mt-DNA in patients with acute kidney injury, often induced by renal IRI (49-51). Moreover, there seems to be a synergistic relationship between donor age and prolonged ischemia in regard to organ damage leading to both functional and morphological deterioration after transplantation as shown in experimental renal transplant models (52).

The potential of Machine Perfusion to preserve older organs

Although CS has been the gold standard preservation method for many years, MP bears critical advantages, allowing for longer preservation times, evaluation of organ quality and the potential of organ reconditioning to maximize the utilization of organs for transplantation (53-56). MP per-se is not novel. As early as at the beginning of the twentieth century, efforts have been made to develop such techniques with the first success achieved by Carrel and Lindbergh, developing a normothermic MP method.(57). Belzer's hypothermic MP technique in the early 1960s then replaced normothermic MP. Nevertheless, CS prevailed and became the gold standard for many years as organs of higher quality had predominantly been utilized without the need for MP (58, 59).

A renaissance of MP then occurred with an augmented demand for the utilization of sub-optimal organs, predominantly those from older and DCD donors. In a first international randomized controlled trial (RCT), 336 consecutive deceased donors were randomly assigned to MP or to CS opening a new era in MP (60). Indeed, hypothermic MP decreased the incidence of DGF in renal transplantation while improving 1-year graft survival rate (61, 62). Moreover, avoiding cold ischemia periods altogether appeared advantageous. Indeed, livers preserved by normothermic MP demonstrated significantly reduced levels of hepatocellular enzyme release, despite a 50% lower rate of organ discard and a 54% longer mean preservation time in a RCT (54). Moreover, normothermic MP was shown to inhibit inflammation and promote graft regeneration (63). RCTs comparing different perfusion techniques are still needed but a clinical trial comparing hypothermic and normothermic MP is already underway in liver transplantation (ClinicalTrials.gov Identifier: NCT04644744).

In addition to improving outcomes, MP facilitates the assessment of organ quality, allowing transplant teams to assess the viability of the graft prior to transplantation (11, 64, 65). Although both hypothermic and normothermic MP seem to be beneficial for transplant outcomes, the advantageous of either approach regarding organ-assessment, and treatment has not been compared yet.

Different markers assessing organ quality have been evaluated, including transaminases, glucose metabolism, lactate clearance, and acid-base balance to access the viability of high-risk livers during normothermic MP (64, 66). In renal transplantation, measuring glutathione S-transferase, lanine-aminopeptidase, N-acetyl-β-D-glucosaminidase, and heart-type fatty acid binding protein during hypothermic MP have been found to be associated with DGF, allowing clinicians to adjust posttransplant recipient management (67). Novel approaches are being developed to further improve the assessment of organs, for example, adding creatinine to the perfusate and calculating creatinine clearance from the perfusate over time (68*).

MP also provides a platform for the administration of therapeutic agents (10, 69, 70) . Experimentally, pharmacotherapy, genetic treatment, mesenchymal stromal cells (MSC), and nanoparticles, have all been successfully administered directly during ex vivo MP (10). For instance, adding human MSCs to porcine kidneys under normothermic MP increases the release of immunomodulatory cytokines including human hepatocyte growth factor, interleukin (IL) −6, and IL-8. Conversely, MSC treatment decreased the release of lactate dehydrogenase and neutrophil gelatinase-associated lipocalin (71). In clinical ex-vivo studies, pairs of DCD kidneys were used, demonstrating ameliorated inflammatory responses and an augmented synthesis of adenosine triphosphate and growth factors, including endothelial growth factor, fibroblast growth factor 2, transforming growth factor α when treated with MSCs during MP. Moreover, normalization of the cytoskeleton and mitosis have been observed in MSC-treated organs. Taken together, the regeneration of human renal tissue ex vivo seems possible, providing a rationale for further clinical application (72). Further support of MP as a platform for organ targeted therapy comes from a recent ex vivo kidney perfusion study providing evidence for an augmented avidity of monoclonal antibodies during ex vivo MP. Administration of CD47-blocking antibody (αCD47Ab), an inhibitor of thrombospondin mediated IRI-signaling during CS resulted in no antibody binding whereas one hour of ex vivo MP, in turn, yielded widely spread abundancy of αCD47Ab through the tissue regions of interest (73). Of clinical relevance, the concept of selectively treating the organ isolated ex-vivo allows for high dosing and cost-effective application while avoiding side effects.

The potential of senolytics

With aging, senescent cells accumulate and tissue regeneration is becoming limited (74-76). Although there is currently not a single marker for senescent cells, they can be characterized by the expression of p21^CIP1 and/or p16^INK4a, DNA segments with chromatin alterations reinforcing senescence, senescence-associated heterochromatin foci, senescence-associated β-galactosidase, and short telomeres (77-80). Of pathophysiological relevance, senescent cells secrete a myriad of pro-inflammatory factors termed the senescence-associated secretory phenotype (SASP) that both disrupt tissue homeostasis, affecting functions of neighbor cells resulting in age-related tissue dysfunction, chronic age-related diseases, and advanced biological aging (81**-84). As senescence represents a cell-autonomous mechanism, preventing the transformation into malignant cells (85), approaches to inhibit cellular senescence by targeting p16^INK4a, Rb, p53, or p21^CIP1 must be well balanced in order to avoid the development of malignancies (86, 87).

Promising evidence exists for the novel drug class of senolytics with the capacity to selectively deplete senescent cells. (69, 88). Several types of potential targets for anti-aging agents have recently been identified in senescent cells, including the BCL family, HSP90 inhibitors, and PI3K/AKT (89-92) (Fig1). Indeed, the accumulation of senescent cells in cardiomyocytes, endothelial cells, smooth muscle cells, cardiac fibroblasts, and cardiac progenitor cells has been successfully decreased by senolytics leading to diminished amounts of SASP, reduced cardiac hypertrophy and decreased size of the left ventricle (93-95). In preclinical studies, senolytics have been able to alleviate age-related dysfunction, inflammation, and disease in multiple organs, potentially making them effective agents if using organs from older donors for transplantation (88). Notably, several clinical trials evaluating the clinical potential of senolytics are currently underway, with first promising results, showing improved physical function in patients with idiopathic pulmonary fibrosis (88, 96). Moreover, another early clinical trial found the treatment with senolytics to reduce senescent cell burden in adipose tissue of diabetic kidney disease patients, further supporting the concept of targeting senescent cells in humans by senolytics (97). In addition, SToMP-AD, an open label pilot phase 1 trial of senolytics for Alzheimer’s disease has been completed (98**, 99).

Figure 1: Mechanisms through which senolytics interfere with aging.

Mechanisms of actions of different senolytics (Dasatinib, Fisetin, Quercetin, Curcumin, PCC1, 17-AAG, 17-DMAG, Geldanamycin, UBC-1325, A-1331852, A1155463) are shown. Interfering with distinct signaling pathways, senolytics facilitate the apoptosis of senescent cells, thereby reducing the secretion of the senescence-associated secretory phenotype. Arrows represent activation; bars represent inhibition; yellow/red particles symbolize the senescence-associated secretory phenotype. Abbreviations: RTK, receptor tyrosine kinase; PI3K, Phosphatidylinositol 3-kinase; Akt, Protein kinase B; HSP90, heat shock protein 90; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; bad, BCL2 associated agonist of cell death; Bax, Bcl-2-associated X protein; Bcl-xL, B-cell lymphoma-extra-large; FoxO4, Forkhead box protein O4; p21, cyclin-dependent kinase inhibitor 1; p53, tumor suppressor p53; PCC1, procyanidin C1; 17-AAG, 17-N-allylamino-17-demethoxygeldanamycin; 17-DMAG, 17-Dimethylaminoethylamino- 17-demethoxygeldanamycin; A-1331852 & A-1155463, both selective Bcl-xL inibitors.

Senolytics may also play a beneficial role in organ transplantation. Experimentally, we have shown that senolytics applied prior to experimental IRI reduced senescent cell burden significantly, alleviated systemic inflammation while reducing levels of cf-mt-DNA, Th17, and IFNγ+ T cells. Moreover, administering senolytics prolonged the survival of old murine cardiac transplants significantly even beyond that of young hearts (22).

Preliminary data have also shown that senolytics can deplete senescent cells from discarded human kidneys perfused by cold MP (unpublished own data). Thus, perfusing old organs with senolytics may help to expand the donor pool and improve outcomes of transplants specifically from older donors.

Conclusion

Old organs have an augmented susceptibility to IRI together with an amplified immunogenicity impacting both short -and long-term transplant outcomes. MP has not only the potential to ameliorate the consequences of IRI but can also serve as a platform for preoperative assessment and organ treatment. Senolytics represent a novel class of agents that deplete senescent cells. Thus far only tested experimentally, senolytics represent a novel approach to improve the quality of older organs, thereby increasing organ availability, and improving transplant outcomes.