Effect of Cellular Senescence in Disease Progression and Transplantation: Immune Cells and Solid Organs

Abstract

Aging of the world population significantly impacts healthcare globally and specifically the field of transplantation. Together with end-organ dysfunction and prolonged immunosuppression, age increases the frequency of comorbid chronic diseases in transplant candidates and recipients, contributing to inferior outcomes. Although, the frequency of death increases with age, limited use of organs from older deceased donors reflects the concerns about organ durability and inadequate function. Cellular senescence (CS) is a hallmark of aging, which occurs in response to a myriad of cellular stressors, leading to activation of signaling cascades that stably arrest cell-cycle progression to prevent tumorigenesis. In aging and chronic conditions, senescent cells accumulate as the immune system’s ability to clear them wanes, which is causally implicated in the progression of chronic diseases, immune dysfunction, organ damage, decreased regenerative capacity, and aging itself. The intimate interplay between senescent cells, their pro-inflammatory secretome, and immune cells results in a positive feedback loop, propagating chronic sterile inflammation and the spread of CS. Hence, senescent cells in organs from older donors trigger recipient’s alloimmune response resulting in the increased risk of graft loss. Eliminating senescent cells or attenuating their inflammatory phenotype is a novel, potential therapeutic target to improve transplant outcomes and expand utilization of organs from older donors. This review focuses on the current knowledge about the impact of CS on circulating immune cells in the context of organ damage and disease progression, discusses the impact of CS on abdominal solid organs that are commonly transplanted and reviews emerging therapies that target CS.

I. Introduction

The fact that the number of individuals over the age of 65 is more than doubling over the next two decades globally carries significant implications for healthcare and for organ transplantation.^1,2 Currently, according to the United Network for Organ Sharing, over 25% of transplant candidates waiting for a heart, lung, liver, or kidney are over 65 years of age. In contrast, only 7% of organs transplanted from deceased donors are in the same age category, primarily due to concerns of inadequate organ function from older donor.³ According to the United States Census Bureau, the projected number of individuals over the age of 65 will surpass the number of individuals under the age of 18 by 2034, which will further exacerbate the problem of aging recipients and donors.⁴ The risk of organ failure increases with age.^5–7 However, multiple analyses of selected older organ recipients demonstrated that advanced age alone is not a contraindication for organ transplantation.^8–12 Still the waitlist percentage of older candidates is not reflective of the disease burden in the population as the frequency of medical comorbidities increases exponentially with age, decreasing the likelihood of successful transplant. Frailty and comorbidities reduce the capacity for older recipients to withstand surgical and immunosuppressive stress, contributing to posttransplant organ dysfunction, infections and malignancies that may be related to the immunosenescence of aged organisms. From an organ availability perspective, the mortality increases with age, but organs from older individuals are infrequently used for transplant reflecting concerns about organ durability and function as it is well-established that age contributes to increased immunogenicity and decreased regenerative capacity of transplant organs, thus limiting their clinical use.^13,14 Chronological age is the greatest risk factor for most common chronic diseases by orders of magnitude compared to commonly treated risk factors.⁵ Deciphering what in aging biology mediates that vulnerability to disease and finding ways to therapeutically intervene in that biology has the potential to transform transplantation medicine, extending the age range of donors and recipients.

II. Aging biology

The goal of therapeutically targeting aging biology to extend the period of health in old age is termed Geroscience.¹⁵ The pursuit of Geroscience led to identification of nine hallmarks of aging biology by experts in the field.^15–17 These include altered intercellular communication, genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence (CS) and stem cell exhaustion. Each hallmark fulfills three distinct criteria: it manifests during normal aging; its aggravation accelerates normal aging; and its amelioration delays normal aging and increases lifespan.¹⁶ Recently, the hallmarks of aging were reclassified in a hierarchy with genomic instability, telomeric attrition, epigenetic changes, loss of proteostasis being primary drivers of aging biology, all reflecting mechanisms of accumulation of macromolecular damage in cells.¹⁷ The antagonistic hallmarks of aging biology are mechanisms that evolved to combat the primary drivers of aging and include CS, changes in mitochondrial function, and altered nutrient sensing. Finally, the integrative hallmarks of aging comprise systemic responses to chronic activation of the antagonistic hallmarks and include stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis. At least two of these integrative hallmarks are known to have a direct impact on the success of transplantation: chronic inflammation and stem cell exhaustion.^18–20

III. Cellular senescence

CS, as an antagonistic hallmark of aging, is a cellular response to a variety of internal and external stressors, including genotoxic, proteostatic, or organelle stress (Figure 1).²¹ A signaling cascade is deployed to stably arrest cell-cycle progression. CS protects the organism from pathological conditions such as tumorigenesis.²² A key feature of senescent cells is a robust secretome of growth factors, proteases, and pro-inflammatory cytokines and chemokines.²¹ This senescence-associated secretory phenotype, or SASP, is involved in tissue remodeling and attraction of immune cells.²³ Hence, CS promotes wound healing, which is resolved by immune clearance of senescent cells from the wound site. Senescent cell abundance increases with chronological age in humans.²⁴ CS is also associated with numerous chronic conditions including obesity, metabolic syndrome, diabetes, pulmonary and hepatic fibrosis, osteoporosis, osteoarthritis, steatosis, chronic kidney disease, cardiovascular disease, immune dysfunction, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s.^17,25–28 In recent years, CS gained much attention as a target for therapeutic interventions aimed at eliminating senescent cells or attenuating their inflammatory phenotype via senolytics and senomorphics, respectively.^29–34

Figure 1. Mechanisms of senescence and characteristics of senescent cells.

Chronic diseases, infectious agents, trauma, immune dysregulation, metabolic disorders, frailty, and psychosocial stress are associated with increased senescent cells. Senescence arises down stream of signaling events in response to a variety of intracellular stressors, including: (1) mitochondrial dysfunction associated with changes in mitochondrial metabolism, morphology, and increase in ROS; (2) DNA damage and epigenetic changes; (3) replicative stress with telomere shortening and damage; and (4) oncogene activation. These activate the DDR, which converges on p53 and/or p16 activation, which drive cell cycle arrest. Prolonged activation of the DDR induces cellular senescence, characterized by, in addition to the above, SAHF, TAF, SAMD with release of mt-DNA, CCF, metabolic changes including increased lysosomal SA-β-gal activity, expression of new proteins on the cell surface (surfaceome) and a SASP that can spread senescence in a paracrine and endocrine fashion to neighboring cells and organs respectively. AMPK, AMP-activated protein kinase; ATP, adenosine triphosphate; CCF, cytoplasmic chromatin fragments; DDR, DNA damage response; EV, extracellular vescicles; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; MAPK, mitogen-activated protein kinase; miRNA, micro RNA; MMP, matrix metalloproteinase; mtDNA, mitochondrial DNA; mTOR, mammalian target of rapamycin; ROS, reactive oxygen species; SA-β-gal, senescence-associated β-galactosidase; SAHF, senescence-associated heterochromatin foci; SAMD, senescence-associated mitochondrial dysfunction; SASP, senescence-associated secretory phenotype; TAF, telomere-associated foci; TCA, tricarboxylic acid; TGF-β, tumor growth factor beta; VEGF, vascular endothelial growth factor.

A growing body of research demonstrates that the features of senescent cells vary depending on their cell lineage and the type of stress that instigated CS.³⁵ Furthermore, the pattern of senescent cells accumulation varies between organs.^16,36,37 Common features of CS include distinct transcriptional changes, upregulation of cell-cycle arrest regulators (p53, p16^INK4a and p21^CIP1), telomere-associate DNA damage foci, senescence-associated distension of satellites, compromised mitochondrial function, and upregulation of lysosomal senescence-associated β-galactosidase (SA-β-gal) activity.^33,38–40 Senescent cells from one organ may affect the entire organism through SASP, which often includes IL-6, IL-8, TNF-α, CCL2, CCL20, and matrix metalloproteases.⁴¹ Senescent immune cells appear to be particularly deleterious, perhaps because of their inherent secretory capacity plus the fact that senescent immune cells contribute to immunosenescence, defined as phenotypic changes of immune cell subtypes, increased memory T cells, diminished ability to mount antigen-specific responses, and persistent low-grade inflammation coined as “inflamm-aging”.^42–45 With aging and in chronic pathological conditions, there is an intimate interrelationship between senescence of immune and nonimmune cells that results in a positive feedback loop, leading to progressive accumulation of senescent cells that propagates inflammation and immune dysregulation, thereby driving further CS and organ damage.⁴⁶

In order to appreciate the complexity of CS, it is crucial to understand the role of senescent immune and nonimmune components as they contribute to organ damage in chronic disease and transplantation (Figure 2). Herein, we attempt to define current knowledge about the impact of CS on circulating immune cells in the context of organ damage and disease progression, discuss the impact of CS on abdominal solid organs that are commonly utilized for transplantation (liver, kidneys), and review emerging therapies that target CS with the aim of optimizing and delaying physiological and pathological decline in organ function.

Figure 2. Impact of senescent cells on the organism and target therapies.

SASP from a variety of senescent cell types can drive healthy, normal cells to senescence resulting in: (1) immunosenescence with accumulation of T-memory cells, decreased in cellular and humoral response, decreased clearance of senescent cells by senescent immune cells, increased inflammation; (2) organ specific damage with renal, hepatic, and endocrine dysfunction, poor neurologic recovery, impaired muscle regeneration, allograft dysfunction; (3) primary disease recurrence (eg, PSC and IgA nephropathy); and (4) chronic disease progression (eg, fibrosis, biliary injury, fat accumulation). In transplantation, ischemia-reperfusion injury and the accumulation of cf-mt-DNA in an older donor causes activation of donor DCs that lead to direct antigen presentation following graft reperfusion and activation of recipient T-cells promoting an alloimmune response and rejection of the transplanted organ. Senotherapies target cellular senescence via senolytics (drugs that target the destruction of senescent cells) and senomorphics (drugs that inhibit the SASP and promote senostasis). cf-mt-DNA, cell-free mitochondrial DNA; DC, dendritic cell; EV, extracellular vescicles; GM-CSF, granulocyte-macrophage colony-stimulating factor; IgA, immunoglobulin A; IL, interleukin; miRNA, micro RNA; MMP, matrix metalloproteinase; mt-DNA, mitochondrial DNA; PSC, primary sclerosing cholangitis; SASP, senescence-associated secretory phenotype; TGF-β, tumor growth factor beta; TLR9, toll-like receptor 9; VEGF, vascular endothelial growth factor.

IMMUNE CELLS

Circulating senescent immune cells contribute to disease progression and organ damage by two distinct mechanisms. First, senescent immune cells drive senescence and damage in solid organs.⁴⁴ Second, senescent immune cells have altered function, reducing their ability to clear senescent cells.^44,47 Healthy immune cells can exacerbate this scenario further. The SASP from senescent cells activates healthy immune cell subpopulations, initiating and propagating an immune response, which can lead to subsequent organ damage.^48,49 In the setting of transplantation, the inciting events that contribute to increased systemic CS are multifactorial, including the age of donors and recipients, the chronic disease state of recipients, ischemia reperfusion injury (IRI) at the time of organ implant, alloimmune response to the donor organ, and primary disease recurrence following transplantation.^28,47,48,50 This is exacerbated substantially further in allogeneic bone marrow transplantation by pretransplant immunoablative therapy.^51,52

I. Preclinical data on senescent immune cells

In mice, deletion of a crucial DNA repair gene, Ercc1, specifically in the hematopoietic system was used to drive primary CS of immune cells.⁴⁴ This was accomplished using the Vav-iCre promoter to drive Cre recombinase expression throughout the immune compartment. CS arose as a consequence of spontaneous endogenous DNA damage that was no longer repaired in the absence of ERCC1, a subunit of a DNA repair endonuclease. Adult Vav-iCre^+/−;Ercc1^-/fl mice demonstrate attrition as well as senescence of immune cell subpopulations. Markers of CS (p16^Ink4a and p21^Cip1 mRNA) and SASP are significantly upregulated in B cells^B220+CD19+, T cells^CD3+, NK cells^CD3-NK1.1+, and macrophages^CD11b+F4/80+.⁴⁴ In addition, the mice show evidence of premature immunosenescence, including T-cell exhaustion, an increase in memory T-cells (CD44⁺), upregulation of PD-1 receptor on CD4⁺ cells, and, as a consequence, impaired cellular and humoral immune responses similar to that of much older wild-type animals.⁴⁴ Adult Vav-iCre^+/−;Ercc1^-/fl animals secondarily show an increase in p16^Ink4a and p21^Cip1 expression, and SA-β-gal activity in parenchymal cells of solid organs, including renal tubule epithelium and hepatocytes. This secondary CS leads to organ damage, e.g., proteinuria, elevated serum transaminases and amylase, and impaired muscle regeneration. Adoptive transfer of splenocytes from the Vav-iCre^+/−; Ercc1^-/fl mice into young adult recipient animals drives CS in the recipients, confirming the role of senescent immune cells in causing secondary CS.⁴⁴ In contrast, transplantation of young splenocytes into an aged recipients reduces expression of CS biomarkers in a variety of organs.⁴⁴ This confirms that senescent immune cells in an older organism lose the capacity to clear senescent cells. Notably, transplantation of splenocytes from old wild-type mice into young recipients is an even more potent driver of CS in trans than immune cells from the transgenic mice.⁴⁴ These preclinical studies illustrate how aged donor and recipient immune systems could contribute to adverse outcomes in transplantation.

In other preclinical studies instigated by the SARS-CoV2 pandemic, it became clear that senescent cells also modulate the response to pathogen-associated molecular patterns (PAMPs) and infection. When challenged with the classical bacterial PAMP, LPS, senescent cells or old mice harboring senescent cells hyper-respond, secreting elevated levels of pro-inflammatory cytokines and chemokines such as IL1α, IL1β, IL6, IL10, and CCL2, relative to nonsenescent cells or young mice, respectively.⁵³ The same occurs upon challenge with a viral coat protein or upon viral infection of naïve, aged mice, including in the liver, lung, and kidney. This contributes to cytokine storm, multi-organ failure, and mortality in old mice.⁵³ Pharmacologic or genetic clearance of senescent cells in old mice improves mortality by ~50%.^53,54 Conversely, SARS-CoV-2 infection drives senescence.⁵⁵ These studies illustrate how CS in aged recipients or donated organs might negatively impact responses to environmental pathogens. What remains unclear is whether immunosuppression would exacerbate or attenuate this hyperinflammatory, tissue damaging state or potentially accelerate CS.

II. Human studies on senescent immune cells

Similar to the observations in murine aging, human humoral and adaptive immune responses evolve with aging and chronic disease. In the SARS-CoV-2 study cited above, COVID-19 patients show increased CS in their airway mucosa, and increased serum SASP. That SASP drives macrophage and platelet activation, complement lysis, senescence of endothelial cells, and neutrophil extracellular trap formation.⁵⁵ An aged microenvironment in the lungs drives transcriptional changes in alveolar macrophages, which prevents their proliferation and a robust response to viral infections.⁵⁶ In chronic kidney disease (CKD), urea levels are correlated with impaired endocytosis and the maturation of monocytes and dendritic cells, resulting in decreased antigen-presentation and maturation of antigen-specific T cells.^57,58 Patients on long-term hemodialysis exhibit evidence of premature aging as reflected by a higher percentage of senescent mononuclear cells with significant telomere attrition, a CD14^high/CD16^low phenotype, a pro-inflammatory cytokine overproduction and upregulation of p53.⁵⁹ In turn, the pro-inflammatory cytokines of SASP promote chronic inflammation and induce CS via paracrine and endocrine mechanisms, promoting tissue damage and potentially tumorigenesis.^60,61 Interestingly, peripheral blood cells from renal transplant recipients demonstrate significantly greater telomere attrition 12 months following transplantation relative to patients on hemodialysis for 12 months or healthy controls.⁶² The shortening of telomeres is especially pronounced in patients treated with mycophenolate mofetil. In another study, older kidney transplant recipients had decreased frequency of naïve CD4+ and CD8+ T cells, an increased frequency of terminally differentiated senescent NK cells, and increased T cells expressing the immunosenescence marker (KLRG1), which was shown to correlate with increased posttransplant infections and impaired immunity to cancer.^63,64 Although, the etiologies of CS differ in the above studies, the accumulation of senescent immune cells in pretransplant and transplant patients contribute to premature aging of organ recipients and the development of age-related diseases. This is a critical area for future research as it is well-documented that the main causes of death among solid organ transplant recipients are infections and age-related chronic conditions such as malignancies and cardiovascular disease.^65,66

III. Immune system response to CS signals

There is also substantial data on the role of nonsenescent immune cells responding to senescent cells, leading to propagation of CS and loss of tissue homeostasis. In a murine model of cardiac allotransplantation, senescent cells in old but not young donors are a critical driver of organ immunogenicity, activation of dendritic cells in recipient animals, and immunologic graft loss.⁴⁸ Clearance of senescent cells from aging hearts prior to transplant dampened age-specific immune response and prolonged survival of allografts.⁴⁸ The authors identified cell-free mitochondrial DNA (cf-mt-DNA) as a potent driver of CS in this model. They also demonstrated that cf-mt-DNA is increased in the serum of older human deceased donors compared to young and activates human dendritic cells ex vivo.⁴⁸

In diabetic kidney disease, M1 pro-inflammatory macrophages accumulate in the glomeruli and induce CS in glomerular endothelia cells by increasing the abundance of reactive oxygen species in humans and a preclinical disease model.⁶⁷ Here, macrophage plasticity should be considered. In the acute injury phase, M2 alternative macrophages are activated by the Th2 type cytokines IL-4 and IL-13, or by M2 phagocytosis of apoptotic cells. This contributes to healing, suppression of inflammation, reduction of immune infiltration, and reduced fibrosis.⁶⁸ However, senescent cells that are associated with chronic inflammation or aging can inhibit the transition from M1 to M2 via their SASP, thereby propagating tissue injury. For example, following IRI, the intrarenal microenvironment of aged mice inhibits the M1 to M2 transition, leading to the accumulation of M1 and fibrosis.⁶⁹

In acute liver injury, the SASP factor, CCL2, from senescent murine hepatocytes activates tissue-resident macrophages leading to TGFβ-mediated senescence transfer to nonsenescent hepatocytes and tissue damage.⁷⁰ Inhibition of this pathway reduces secondary senescence and enhances liver regeneration. Notably, CCL2 was identified as the SASP factor that most consistently reports biological (rather than chronological) age in mice and is increased in frail humans.⁷¹ On the other hand, SASP potentiates CD4+T-cell-mediated clearance of senescent premalignant hepatocytes with assistance of tissue-resident monocytes/macrophages.⁷²

Although while young and healthy, CS maintains body homeostasis by promoting regeneration and cancer surveillance, aging and chronic conditions such as obesity, metabolic disorders, cardiovascular disease, CKD and transplantation, promote accelerated accumulation of senescent cells. Senescent cells, whether immune or otherwise, drive chronic sterile inflammation and immunosenescence, which contributes to failure to clear senescent cells. This, in turn, promotes tissue damage and further immune dysfunction. Better understanding of precisely how CS perturbs immune function and promotes inflammation would be valuable for designing novel therapies to improve transplant outcomes.

LIVER

The complex network of hepatic functions includes metabolism, detoxification, coagulation, and immune response. This requires multiple cell types that interact in a spatiotemporal manner to ensure normal function of the organ and the homeostasis of the entire organism.⁷³ Recent single-cell RNA-sequencing efforts provided a detailed human liver atlas that describes over a dozen major types of liver cells.⁷⁴ Of those, five subtypes, hepatocytes, hepatic stellate cells (HSCs), Kupffer cells, liver sinusoidal endothelial cells (LSECs) and cholangiocytes comprise the majority of hepatic parenchyma with hepatocytes contributing to 70% and HSCs, Kupffer cells, LSECs, cholangiocytes contributing to approximately 30% of the liver.^73,75

I. Senescent hepatocytes

To date, the majority of liver aging biology research has focused on hepatocytes.³⁷ CS can be driven by telomere attrition due to replication and cell division or by other types of stress including infection, toxins, oxidative stress, genotoxic entities, ischemia reperfusion, or oncogenic stress.⁷⁶ Hepatocytes and cholangiocytes do not undergo replicative senescence in humans despite the impressive regenerative capacity of the liver.^77,78 This is in part due to a subset of hepatocytes that express telomerase, enabling telomere lengthening and extended replicative capacity.⁷⁹ In contrast, with healthy aging, the number of polyploid hepatocytes accumulate with time, which limits their replicative capacity, thus defining those cells as senescent.⁸⁰

In the setting of pathologic conditions, CS provides protection by blocking proliferation of damaged cells early in the disease. However, as disease progresses, there is an accumulation of senescent hepatocytes characterized by increased expression of cell cycle inhibitors p53/p21^CIP1 and p16^INK4a, larger nuclear area, γH2AX (a marker of DNA damage) and SASP factors that promote the conversion of nonsenescent cells to senescent phenotype in a paracrine manner.^28,41,81 In contrast to healthy liver, CS, loss of telomerase expression and loss of regenerative capacity are characteristics of progression from fibrotic disease to cirrhosis.⁸² Gain of function mutations at the promoter of TERT reintroduces telomerase expression and enables the development of hepatocellular carcinoma in cirrhosis.⁸²

II. Senescent cholangiocytes

As with senescent hepatocytes in pathological conditions, senescent cholangiocytes are characterized by p21^WAF1 and p16^INK4a overexpression, telomere-shortening, secretion of SASP factors as well as increased SA-β-gal activity. Senescent cholangiocytes are increased in the biliary systems of patients with primary sclerosing cholangitis (PSC) and primary biliary cholangitis.⁸³ Senolysis reduces fibrosis in a murine model of PSC. The SASP of senescent cholangiocytes spreads senescence to bystander cholangiocytes and activates the innate and adaptive immune responses that subsequently leads to the autoimmune response and destruction of the biliary system.^84,85 Further, as senescence spreads in the injured ducts, it impairs the removal and replacement of senescent cholangiocytes exacerbating inflammation and loss of bile ducts.⁸⁵ As in other pathologies, senescent cholangiocytes, characterized by overexpression of p21^WAF1/CIP1, are increased in the setting of rejection following liver transplantation and decreased in response to treatment of rejection.⁸⁶

III. Senescence of other liver cell types

In contrast to hepatocytes and cholangiocytes, HSCs and Kupffer cells demonstrate evidence of replicative senescence as their telomeres undergo shortening in vivo and in vitro.^21,78,87 However, the key role of senescence in relation to HSCs involves the regulation of extracellular matrix (ECM) deposition. During liver injury, HSCs acquire a myofibroblastic phenotype and increased secretion of ECM that subsequently leads to fibrosis.⁸⁸ The induction of senescence in activated HSCs limits their fibrogenic response in the setting of tissue damage by decreasing ECM production and promoting clearance of activated/senescent HSCs by immune cells.⁸⁹ In the absence of injury, HSCs undergo a milder form of activation characterized by proliferation stimulated by the HSC-related growth factor (PDGFRβ), accumulation of intracellular lipid droplets, an increase in expression of alpha smooth muscle actin (αSMA), collagen 1α1, collagen 1α2, and p-moesin as well as alteration in expression of matrix metalloproteinases and their inhibitors that affect deposition and content of the ECM.⁹⁰ Interestingly, HSCs from aged livers become hyperresponsive in the setting of injury with overproduction of ECM components leading to architectural distortion, elevated hepatic vascular tone, and in turn exacerbation of liver injury and fibrosis, which may reflect a role of CS.⁹¹ These findings are relevant not only for aging patients with primary liver disease but also in the setting of liver transplantation of organs from older donors, as well as the recovery of older live donors following partial liver donation.

LSECs provide a critical contact point between portal circulation and the liver serving the function of bidirectional transfer of metabolites, endocytosis, immunotolerance, and maintenance of hepatic microenvironment.⁹² Senescent LSECs have elevated p16^INK4a expression, evidence of mitochondrial stress, and increased pro-inflammatory markers. This phenotype becomes more prevalent in aging and diseased liver leading to defenestration and reduction of porosity in the sinusoidal endothelium, thus preventing exchange of plasma and plasma proteins.^93,94 Decreased sinusoidal permeability triggers a compensatory upregulation of scavenger receptors on the LSECs and promotes uptake of toxic substances, which in turn further promotes senescent phenotypes.⁹⁴ Ultimately, this leads to increased oxidative stress in liver sinusoids and extrahepatic vasculature as well as LSEC thickening and fibrosis.^95,96

Although, Kupffer cells represent the largest population of tissue-resident macrophages in the body, the impact of senescence on these cells is understudied.⁹⁷ Based on the study of macrophages in other tissues, it is known that aging leads to a reduction in autophagy and phagocytosis, secretion of pro-inflammatory mediators, and alteration of cell morphology.⁹⁸ As discussed in a prior section, Kupffer cells also act in the role of responders to the aging microenvironment and other senescent cell subpopulations. CS of nonimmune cells contributes to this pro-inflammatory microenvironment, which induces Kupffer cell polarization promoting an M1 proinflammatory phenotype implicated in a number of pathologic liver conditions including fibrosis.^98,99

KIDNEY

The complex network of renal functions includes electrolyte, fluid, and acid-base balance, filtration of toxins and waste, hormone production, and regulation of hemodynamics. These diverse functions necessitate a large number of highly specialized cells that include over 16 epithelial subtypes in addition to a significant number of endothelial, immune, and interstitial cell types.¹⁰⁰ In aging and pathophysiologic conditions, senescence impacts diverse renal cell subpopulations and compartments with the accumulation of senescent cells as a common feature of the aging kidney and CKD.^101–106 This is not surprising as both aging and CKD have similar clinical and pathological presentation. From a clinical standpoint, both manifest with proteinuria, reduced glomerular filtration rate, disruption of electrolyte homeostasis, and fluid retention. Pathohistological findings of the aging kidney and CKD include podocyte loss/dysfunction, glomerular basement membrane thickening, glomerulosclerosis, decline in nephron number/size, tubular atrophy, fibrosis, inflammation, and loss of vascular compartment.¹⁰⁷ Eventually both processes render renal vulnerability to toxic or hemodynamic stress and limited regenerative capacity.

I. Senescent renal tubule epithelial cells

The tubular epithelium is the major source of senescent cells in CKD.¹⁰⁸ Chronic oxidative, toxic, and metabolic stress induce CS in proximal tubular epithelium. Prolonged injury leads to persistent SASP, inflammation, and the secretion of profibrotic factors.¹⁰⁷ For example, indoxyl sulfate, a byproduct of metabolism, induces CS in proximal tubules by upregulating reactive oxygen species and the p53 pathway, suppressing cell proliferation and increasing TGF-β1 and α-SMA, which eventually leads to fibrosis.¹⁰⁹ Aging- and uremia-associated oxidative stress leads to cytotoxic DNA damage.^110,111 In acute kidney injury, CS of tubular epithelial cells is induced via components of the innate immune system, the Toll-like and interleukin 1 receptors, and leads to persistent inflammation, damage to renal tubules, and fibrosis.¹¹² This is relevant in many aspects of transplantation including delayed graft function, rejection, and infection.

Although proximal tubular epithelium is thought to be the main site of senescent cells, different stressors target diverse cell populations, which impacts the localization and type of CS in the kidney. For example, p16^INK4a is significantly upregulated in glomerular and interstitial cell nuclei in patients with glomerular disease compared to normal controls or patients with tubulointerstitial nephritis. The expression of p16^INK4a in tubular cytoplasm is higher in kidneys from patients with proteinuria, fibrosis, and atrophy.¹⁰¹ Of note in glomerular disease, podocyte-derived TGF-β1 induces nuclear translocation of p16^INK4a in glomerular endothelial cells, leading to endothelial senescence and disease progression.¹¹³ Hypertension also contributes to renal CS. In several preclinical models of hypertension, including aldosterone and angiotensin II induced disease, upregulation of SA-β-gal activity and p21^Cip1 expression in proximal tubular cells is observed, which contributes to reduced regenerative capacity of tubular endothelium.^114,115 Further, in a setting of hypertension, telomere shortening is observed in vascular endothelium.¹¹⁶

II. Senescent glomerular endothelial cells

Other contributors to senescence-associated renal dysfunction are senescent glomerular endothelial cells. Senescent endothelial cells modulate podocyte function through the SASP factor, plasminogen activator inhibitor 1 (PAI-1).¹¹⁷ Inhibition of PAI-1 prevents glomerulosclerosis and podocyte loss in aging animals. In human renal transplantation from elderly donors, PAI-1 expression in senescent endothelium is predictive of poor outcomes.¹¹⁷

Loss of Klotho, an anti-aging factor highly expressed in the kidney, is implicated as a driver of medial vascular calcifications and progression to CKD.¹¹⁸ Klotho-deficient mice demonstrate a progeroid phenotype and tissue-specific age-associated defects.¹¹⁹ Similarly, patients with CKD lack Klotho, further implicating its critical role in renal homeostasis.¹¹⁸ Although, the exact mechanisms are not known, uremic toxins are thought to induce Klotho gene hypermethylation and downregulation of its expression.¹²⁰ Several studies suggest that Klotho acts systemically to prevent aging phenotypes by decreasing oxidative stress through upregulation of superoxide dismutase, inhibition of endothelial senescence, antifibrotic properties, suppression of the insulin-IGF-1 signaling pathway that in turn is associated with life extension, as well as by systemic regulation of mineral metabolism.^111,121 The therapeutic potential of Klotho should be further explored as its systemic delivery has been shown to ameliorate the aging phenotype in klotho mutant mice.¹²²

III. Renal CS and transplantation

In transplantation, there is evidence that CS is correlated with the biological age of the organ and posttransplant function. In the study of human renal allografts, serum creatinine at 6-months posttransplant and subsequent graft dysfunction strongly correlate with donor age and the senescence marker, p16^INK4a, on pretransplant graft biopsy.¹²³ Similarly, p16^INK4a levels, in combination with chronological donor age, was the best predictor of renal allograft function at 1 year following transplantation. In another study, p16^INK4a and p27^KIP1 were upregulated in glomeruli, renal tubules, and interstitial cells of kidney allografts with evidence of chronic rejection and in kidneys from older donors when compared to healthy or younger individuals.¹⁰²

At the time of implant, CS is increased as a result of IRI causing oxidative stress and overexpression of p16^INK4a and p21^CIP1.¹²⁴ However, the temporary induction of cell cycle arrest via the p21^CIP1 pathway may be advantageous by allowing time for DNA damage repair and has been shown to be beneficial in the setting of IRI, whereas prolonged cell cycle arrest via p16^INK4a and senescence contributes to inferior transplant outcomes in animal models.¹²⁴ In patients with immunoglobulin A nephropathy, markers of CS (increased SA-β-gal activity, p16^INK4a and p21^CIP1 expression) are significantly elevated in tubular epithelial cells compared to healthy controls, and the increase in these markers correlates with clinical and pathological disease progression.¹²⁵ These observations are important for management of primary disease and primary disease recurrence following kidney transplantation.

EMERGING THERAPIES

Given the central role of CS in aging and pathogenesis, it is not surprising that CS emerged as a promising new target for therapies, with the coining of a new term “senotherapy”. Senolytics selectively deplete senescent cells, whereas senomorphics suppress markers of senescence or their SASP.¹²⁶ Senolytics offer some therapeutic advantage as they clear senescent cells, eliminating SASP and require intermittent rather than chronic administration.¹²⁷ In contrast, senomorphics may prove more advantageous as they do not eliminate acute CS required for wound healing.¹²⁸

Over a decade ago, the first proof-of-concept animal study demonstrated causal relationship between SC and aging.¹²⁹ Life-long removal of p16^Ink4a-positive senescent cells in the BubR1 progeroid mouse delayed onset of age-related pathologies in these animals and late-life clearance delayed the progression of already acquired age-related disorders.¹²⁹ This study paved the way for discovery of therapies to target pro-aging and pro-pathologic effects of CS. However, the development of new therapies is hampered by the need to preserve beneficial effects of SC such as tumor suppression and tissue regeneration, avoid potential drug-related toxicities e.g., hyperglycemia, thrombocytopenia, immunosuppression, and the need for tissue-specific targets due to differential tissue vulnerabilities and senescence phenotypes.^127,130,131

Transplant candidates and recipients exhibit evidence of premature aging and increased rate of age-related co-morbidities by virtue of suffering from chronic end-stage organ disease and subsequently being exposed to life-long immunosuppression, leading to increased incidence of metabolic and cardiovascular disease, renal dysfunction, infections and cancer.^63,132–139 As discussed earlier, the accumulating evidence suggest that CS plays a causal role in the development and progression of these conditions. Therefore, a number of senotherapeutic agents have been tested in the preclinical setting and are slowly introduced into clinical trials to combat age-related diseases (Table 1).

Table 1.

Senotherapy clinical trials with focus on aging, frailty, and age-related conditions.

Senolytics
Drug	Indication	Phase	Trial registration number
Fisetin PI3K/Akt pathway inhibitor	Frailty	2	NCT03675724, NCT03430037, NCT04733534,
	Healthy participants	2	NCT04313634, NCT04313634
Luteolin ROS, PGE2, COX2 inhibitor	Memory in healthy aging		NCT04468854
Quercetin PI3K/Akt pathway inhibitor	Age-related macular degeneration	2	NCT05062486
Dasatinib and quercetin Tyrosine kinase inhibitor and PI3K/Akt pathway	Alzheimer’s	1 and 2	NCT04063124, NCT04785300
		2	NCT04685590
	Aging	1 and 2	NCT05422885
		2	NCT04946383, NCT05838560, NCT04313634
	Diabetic nephropathy	2	NCT02848131
	Frailty	2	NCT04733534
	Healthy participants	2	NCT04313634
	Liver disease	1 and 2	NCT05506488
	Idiopathic pulmonary fibrosis	1	NCT02874989
UBX1325 Bcl family	Age-related macular degeneration/diabetic macular edema	1	NCT04537884
		2	NCT05275205, NCT04857996
UBX0101 P53/MDM2 interaction inhibitor	Osteoarthritis	NA	NCT04349956
		1	NCT03513016
		2	NCT04229225, NCT04129944
Navitoclax Bcl family	Liver disease	1	NCT02143401
Catechins Bax/Bcl-2, Nrf2, and PI3K/Akt/mTOR pathways	Liver disease	1	NCT03278925
SASP inhibitor
MitoQ Antioxidant, inducer of superoxide dismutase and glutathione peroxidase	Multiple sclerosis	1 and 2	NCT04267926
	Aging	1	NCT05686967
Ganoderma lucidum Nrf2, mTOR, and MAPK pathways	Cardiometabolic syndrome	2	NCT05079529
	Parkinson disease	3	NCT03594656
Equol Nrf2/ARE pathway	Cognition in healthy volunteers	2	NCT05741060
Melatonin Keap1/Nrf2/ARE pathway and SIRT1	Healthy	NA	NCT05419466
		4	NCT04831398
	Aging	NA	NCT03954899
	Stroke	NA	NCT03843008
		3	NCT05857046
		4	NCT05247125
	Multiple sclerosis	1	NCT03498131
		1 and 2	NCT03540485
	Encephalopathy	1	NCT02621944
	Wound healing	2	NCT05122130
	Coronary artery disease	NA	NCT05552586
	Obesity	NA	NCT05759429
	Cardiovascular disease	NA	NCT04631341
		2	NCT05257291
	Renal disease	3	NCT05084196, NCT05570526
	Osteoarthritis	NA	NCT04795336
	Osteopenia, osteoporosis	1	NCT04233112
		4	NCT04864509
Alpha-ketoglutarate Intermediate of tricarboxylic acid (TCA) cycle, imitates caloric restriction	Healthy aging	2	NCT05706389
Rapamycin mTOR signaling pathway	Ovarian aging	2	NCT05836025
	Tuberous sclerosis	3	NCT05534672
	Muscle aging	NA	NCT05414292
	Otolaryngologic disease	2	NCT05342519
	Amyotrophic lateral sclerosis	2	NCT03359538
	Aging, longevity	1	NCT04994561, NCT04742777, NCT04608448
		2	NCT04488601, NCT02874924
	Age-related macular degeneration	2	NCT00766337
	Alzheimer’s disease	2	NCT04629495
	Insulin sensitivity	NA	NCT05233722
	Cardiac function in aging frail adults	1	NCT04996719, NCT01649960
	Sarcopenia	1	NCT00891696
	Pompe disease	1	NCT02240407
Sirolimus mTOR signaling pathway	Aging	2	NCT05237687
Metformin AMPK and NF-κB pathways	Aging, longevity	1	NCT04994561, NCT02308228, NCT03996538, NCT03713801, NCT03107884, NCT04994561, NCT03072485
		3	NCT04264897, NCT03309007, NCT04221750, NCT03861767
		4	NCT02432287, NCT02745886
	Age-related macular degeneration	2	NCT02684578
	Frailty	2	NCT03451006
	Cognitive impairment	2	NCT00620191
		4	NCT02409238
	Dementia	2	NCT01965756
	Neuromuscular disease	3	NCT05532813, NCT01995032
	Visual impairment	2	NCT03490019
	Depression	4	NCT00834652
	Healthy	1	NCT03772964
Resveratrol SIRT1/NF-κB pathway	Friedreich ataxia	2	NCT03933163
	Peripheral artery disease	3	NCT03743636
	Amyotrophic lateral sclerosis	2	NCT04654689
	Age-related macular degeneration	2	NCT05062486
Curcumin AMPK, sirtuin, PI3K/Akt, NF-κB, and Nrf2 pathways	Healthy	NA	NCT05414838, NCT05775237
		1	NCT05334043
	Alzheimer’s disease	1 and 2	NCT05774704
	Amyotrophic lateral sclerosis	2	NCT04654689
	Kidney disease	NA	NCT04413266
		2	NCT03223883, NCT05714670
		3	NCT05613361, NCT05627843
	Chronic conditions in kidney transplant recipients	NA	NCT03935958
	Diabetes mellitus	2	NCT05753436
		1 and 2	NCT05407467
	Wound	NA	NCT05819632
		1	NCT05610878
	Age-related macular degeneration	2	NCT05062486
Spermidine Autophagy	Hypertension	3	NCT04405388

AMPK, AMP-activated protein kinase; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; NA, not applicable; NCT, National Clinical Trial; NF-κB, nuclear factor kappa B; SASP, senescence-associated secretory phenotype.

Applications of senotherapeutics in treatment of metabolic diseases have been widely studied in preclinical models. Administration of ruxolitinim (a Janus kinase 1 or 2 inhibitor) and senolytic cocktail of dasatinib (D) (tyrosine kinase inhibitor) and quercetin (Q) (kinase/antioxidant) to aged animals successfully ameliorates senescent cells, mitigates lipodystrophy, enhances insulin sensitivity and glucose tolerance, lowers circulating inflammatory mediators, prevents migration of monocytes, decreases the number of macrophages in visceral fat, and reduces frailty.^140–142 Similarly, in human trials, senescent cells in subcutaneous fat are associated with altered glycemic control and are cleared within 11 days following the administration of the D+Q cocktail in patients with diabetic kidney disease, although the impact of treatment on renal function and long-term glycemic control is not known.^143–145 Further, the administration of the senolytic cocktail improved cardiac and renal function of aged animals, and decreased hepatic steatosis in subsequent study, albeit, it failed to prevent progression of steatosis in animals with nonalcoholic liver disease-induced hepatocellular carcinoma.^{29,30,141,146} In murine models of cardiovascular disease, BCL2/Bcl-xL inhibitor reduces cardiac hypertrophy and fibrosis with compensatory regeneration in cardiomyocytes and rescues the functional decrease in ejection fraction following myocardial infarction, whereas D+Q reduces CS and plaque calcifications in a model of atherosclerosis.^147–149

Frailty, loss of muscle mass and strength are common in transplant candidates and recipients, thus it is very intriguing and promising that a BCL2/Bcl-xL inhibitor reduces the loss of body weight, improves strength, and increases muscle regeneration in a rat model of Duchenne muscular dystrophy.¹⁵⁰ Similarly D+Q improves muscle function in mice following the irradiation-induced senescence.³⁴ The first-in-human, open-label clinical trial of D+Q in patients with idiopathic pulmonary fibrosis, a cellular senescence-driven disease, demonstrated improvement in physical function and scores in the Short Physical Performance Battery 5 days after 3-week course of D+Q therapy.^151,152

Further, it has been demonstrated that frail transplant candidates have lower cognitive scores than nonfrail and have impaired cognitive recovery following transplantation.¹⁵³ Although, clinical trials on the use of senolytics in neurodegenerative conditions are in progress (NCTO4063124), it is known that overexpression of senescence markers in variable regions of the brain are associated with neurodegenerative diseases in humans.^154–156 Clearance of CS in healthy aged mice and neurodegenerative models with BCL2/Bcl-xL inhibitor or D+Q cocktail enhances brain function, reduces neuroinflammation and accumulation of pathologic markers and improves cognitive deficit in those animals.^157–159

The impact of senolytics on the immune system is variable. Administration of BCL2/Bcl-xL inhibitor (senolytic and anti-cancer compound) triggers premature cell death of noncancerous cells infected with influenza A virus leading to dysregulation of pro-inflammatory and anti-viral cytokines, which contributes to an inability of the immune system to clear the virus with subsequent mortality of infected animals.¹⁶⁰ In contrast, clearance of senescent cells with fisetin (PI3K/AKT pathway inhibitor) or D+Q in acutely infected aged mice with murine-β-coronavirus (related to SARS-CoV-2) reduces CS, inflammation, increases anti-viral response, and reduces animal mortality from 100% to 50%.⁵³ As of now, it is unclear what will be the direct impact of senotherapeutics on transplant recipients, although, it will likely vary based on mechanism of action. For instance, senomorphic-like agent Rapamycin (mTOR inhibitor) extends lifespan in diverse species and is commonly used in transplant to prevent rejection.^161–163 Due to its anti-tumorigenic properties, the drug is utilized in cancer patients as well as transplant patients with a history of malignancy. However, its deleterious impact on the immune system contributes to a high rate of infections and poor healing.¹⁶⁴

To date, the majority of clinical studies focus on safety and tolerability of senotherapy in patients and only few report on efficacy.^165,166 However, senotherapy provides an opportunity to rejuvenate organs from older donors that are currently underutilized and have a potential to eliminate the gap that exists between organ demand and supply.³ Although, the majority of deaths occur among older individuals, the reluctance of the transplant community to use organs from older donors reflects the concerns that aging is associated with decline in organ function and durability. Moreover, experimental and clinical models demonstrate the increased immunogenicity and graft loss of organs from older donors.^48,167,168 Indeed, the transplantation of senescent cells into healthy mice drives frailty, which raises a concern of transferring senescence with organs from older donors during transplantation.¹⁴²

APPLICATION OF EMERGING THERAPIES TO TRANSPLANTATION

Several experimental strategies using senotherapy have been deployed to optimize organs from older donors for transplantation in preclinical models. Pretreatment of older animals with D+Q cocktail reduces systemic levels of cf-mt-DNA and senescent cells in murine kidneys pre-IRI and decreases circulating levels of pro-inflammatory T-cells after IRI.⁴⁸ Further, pretreatment of older donors prior to cardiac procurement prolongs cardiac allograft survival in mice particularly in the presence of clinically relevant immunosuppression protocol with the co-stimulatory CTLA4-Ig blockade that dampens T-cell allo-response.⁴⁸ In both cases, senescent cells are key sources of cf-mt-DNA which is released in the setting of IRI promoting activation of dendritic cells with subsequent initiation of Th1/Th17 alloimmune response. Herein, clearance of senescent cells results in dampening of the immune response and reduces injury of the organ.⁴⁸ In adult mice, p21^Cip1, p16^Ink4a, and p19^Arf senescence markers are upregulated in multiple cell types when the regenerative capacity of the liver decreases.¹⁶⁹ The pretreatment of adult animals with an BCL2/Bcl-xL inhibitor prior to partial hepatectomy reduces p21^Cip1 expression, reduces levels of transaminases in the blood, decreases histological damage and increases liver regeneration by day 7 following partial hepatectomy.¹⁶⁹ These findings are of clinical relevance for donors and recipients during living donor liver transplantation where the sizes of liver remnant and graft are critical to the recovery of both patients and in the setting of split-liver deceased donor transplantation.

Overall, there are a myriad of opportunities to exploit senotherapies in transplantation from stabilizing potential recipient while waiting for an organ, optimizing recipients following transplantation, improving the quality of organs by pretreatment of older donors with senotherapeutics prior to the donation, improving organ regeneration in older living donors and recipients, and optimizing marginal allografts (liver, kidney, heart, lung) on ex vivo perfusion devices with senotherapeutic treatment prior to transplantation thus prolonging ex vivo and in vivo lifespan of donated organs (Figure 3).^31,170

Figure 3. Senotherapies in transplantation.

Clearance of senescent cells and inhibition of senescence-associated secretory phenotype may facilitate reduction in systemic and organ-specific inflammation thus improve metabolism and function of native and transplanted organs, decrease immunogenicity, reverse pathologies in ex vivo perfused organs (eg, steatosis or fibrosis), promote organ regeneration, decrease frailty-associated morbidity in transplant candidates and recipients, decrease primary disease recurrence in transplanted organs, decrease allograft rejection and extend the age of donors and recipients.

In summary, CS of immune and nonimmune compartments play a major role in chronic disease and aging. Manipulating CS to improve outcomes in transplant candidates and recipients by optimizing their overall medical condition and quality of transplanted organs is an emerging field of research. A number of clinical trials on applications of senotherapeutics for chronic conditions are ongoing (Table S1). However, specific use of these therapies in the setting of transplantation and rejuvenation of donor organs need to be explored. D+Q cocktail and BCL2/Bcl-xL inhibitors seem to be promising as therapeutic agents. However, the timing and length of administration, target measurements for success/endpoint of therapies as well as safety profile using senotherapeutics in patients on multi-drug regimens remain to be determined.

Abbreviations Page

αSMA

alpha smooth muscle actin

BCL2/Bcl-xL

B-cell lymphoma 2/B-cell lymphoma-extra large

CCF

cytoplasmic chromatin fragments

CCL2

chemokines C–C motif chemokine ligand 2

CCL20

chemokines C–C motif chemokine ligand 20

cf-mt-DNA

cell-free mitochondrial DNA

CKD

chronic kidney disease

CS

cellular senescence

D

dasatinib

DC

dendritic cells

DDR

DNA damage response

DNA

deoxyribonucleic acid

ECM

extracellular matrix

Ercc1

excision repair cross-complementation 1

EV

extracellular vescicles

HSC

hepatic stellate cells

IRI

ischemia reperfusion injury

IL-6

interleukin 6

IL-8

interleukin 8

KLRG1

killer cell lectin-like receptor G1

LSECs

liver sinusoidal endothelial cells

M1

pro-inflammatory macrophages

M2

alternative macrophages

MMPs

matrix metalloproteinases

mt-DNA

mitochondrial DNA

mRNA

messenger ribonucleic acid

mTOR

mammalian target of rapamycin

NK

natural killer

PAI-1

plasminogen activator inhibitor 1

PD-1

programmed cell death 1

PSC

primary sclerosing cholangitis

ROS

reactive oxygen species

SA-β-gal

senescence-associated β-galactosidase

SAHF

senescence-associated heterochromatin foci

SAMD

senescence-associated mitochondrial dysfunction

SASP

senescence-associated secretory phenotype

TAF

telomere-associated foci

TERT

telomerase reverse transcriptase

TGFβ

tumor growth factor beta

TNF-α

tumor necrosis factor alpha

Q

quercetin