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University of Groningen

Cellular Stress in Aging and Cancer

Sturmlechner, Ines

DOI:

10.33612/diss.170212168

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2021

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Sturmlechner, I. (2021). Cellular Stress in Aging and Cancer. University of Groningen.

https://doi.org/10.33612/diss.170212168

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CHAPTER 2

Cellular senescence in renal ageing and disease

Ines Sturmlechner

Matej Durik

Cynthia J. Sieben

Darren J. Baker

Jan M. van Deursen

Nature Reviews Nephrology, 2017 Feb;13(2):77-89.

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Cellular senescence in renal ageing and disease

Ines Sturmlechner1,2, Matej Durik1, Cynthia J. Sieben3, Darren J. Baker1,3,* and Jan M. van Deursen1,,*

1Departments of Pediatric and Adolescent Medicine, Mayo Clinic, 200 First Street SW, Rochester, Minnesota, 55905, USA. 2Department of Pediatrics, Molecular Genetics Section, University of Groningen, University Medical Center Groningen, Antonius

Deusinglaan 1, 9713 AV Groningen, The Netherlands. 3Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First

Street SW, Rochester, Minnesota, 55905, USA. *Correspondence to D.J.B. and J.M.v.D. [email protected]; [email protected]

The senescence program is implicated in diverse biological processes, including embryogenesis, tissue regeneration and repair, tumorigenesis, and ageing and age-associated diseases. Although studies of in vivo senescence are in their infancy, it is becoming increasingly clear that senescent cells are a highly complex and diverse class of cells, with cell type, senescence-inducing stressor, and senescent cell evolution constituting some of the compounding factors. Senescent cells can be divided into two broad categories, referred to as acute and chronic. Acute senescence is beneficial and consists of a coordinated or programmed cycle of senescent cell generation, signaling, and subsequent elimination. In contrast, chronic senescent cells are thought to arise more slowly from cumulative stresses and seem subject to inefficient elimination, leading to accumulation and deleterious effects through a secretory phenotype. Senescent cells have been identified in many tissues and organs, including kidney, where evidence suggests that both acute and chronic cells play a prominent role in multiple processes. Here, we review the identity, pathways, and biological consequences of senescent cells in kidney development, homeostasis, and pathology. Also, we discuss how senotherapy, or targeting of senescent cells, may be exploited to improve renal function with normal ageing, disease, and therapy-induced damage.

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Chapter 2 Key points

• Cellular senescence is a multi-faceted program implicated in diverse physiological and pathological processes including embryonic development, regeneration and repair, cancer-protection, ageing, and disease.

• Senescent cells that are transiently present (acute senescence) are beneficial, whereas prolonged senescent cell signaling and aberrant accumulation (chronic senescence) impair organ function and promotes pathologies, including the kidney.

• Chronic senescent cells accumulate in the kidneys during natural ageing, and have been causally linked to age-related decline in renal function.

• Senescent cell accumulation also occurs in kidneys in association with several renal diseases and therapeutic damage, and correlates with disease progression or deterioration in several instances. • Therapeutic interventions that target senescent cells, termed senotherapies, have the exciting potential to attenuate age-related renal dysfunction, improve disease outcome, and ensure kidney transplant success.

• Development of effective and safe senotherapies should greatly benefit from future research aimed at understanding of the locations, origins and properties of senescent cells in greater detail. Introduction

Ageing is the progressive decline in tissue function over time, resulting in loss of homeostasis and ultimately organismal fitness4, 5. Although the mechanisms underlying the ageing process have been studied for

almost a century, the reasons why and how we age remain largely elusive. The antagonistic pleiotropy theory of ageing hypothesizes that evolutionarily selected traits ensuring fitness early in life can be detrimental at later ages7. Cellular senescence, a cellular program with permanent cell cycle arrest fits into

this model, where acutely generated senescent cells early in life provide an advantage during the process of development9, 10, tissue regeneration11, and by inhibiting neoplastic transformation12; but aberrant,

chronic accumulation of senescent cells late in life drives various features of ageing, including age-related disease and tissue deterioration13, 14.

Compelling evidence suggests that the distinctive secretome acquired by senescent cells, termed the senescence-associated secretory phenotype (SASP), is a key determinant in attraction, activation, and differentiation of immune cells that results in senescent cell clearance during a process called immune surveillance15-19. This closed cycle of senescent cell generation, signaling, and elimination is usually a

transient, highly efficient, and beneficial physiological process. However, with increased age or under certain circumstances, immune system-mediated removal of senescent cells may be impaired or less efficient, causing pathologies and reduced health and lifespan20.

The kidney consists of a variety of cell types facing unique environments, stressors, and challenges. Studies in cultured cells, mouse models, and human samples, suggest that the senescence program is active over the entire lifetime of a kidney. In this review, we discuss developmental, regenerative,

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cancer-associated, and chronic senescence from a kidney perspective. We discuss the strength of the current evidence for each mechanism in renal development, injury and disease, and areas that would benefit from further research. Finally, we discuss the feasibility and potential therapeutic options for targeting senescent cells, termed senotherapies, to maintain renal function into old age, ameliorate disease, and improve success of renal transplantation.

Features of cellular senescence

Cellular senescence is complex and diverse. It can be induced by a broad spectrum of stressors and in many different cell types, tissues and organs. Also, there is evidence that senescent cells are constantly evolving resulting in substantial variability and heterogeneity of senescent cells even in the same tissue22, 23. Such variability in features makes it difficult to postulate a universally accepted definition of cellular

senescence. However, there seems to be consensus in the field that particular phenotypes and signaling pathways are integral parts of the senescence program (Box 1). The specific “senescence-inducing” signaling events can differ between senescence programs (Figure 1). For example, senescence during embryonic development is dependent on p21Cip1 and does not show signs of a DNA damage response

(DDR)9, suggesting that developmental senescence is a distinctly regulated signaling program and not the

consequence of cumulative cellular stress9. On the other hand, regenerative, cancer-protective, and chronic

senescence often involve induction of p16Ink4a, activation of a DDR and other key molecules, including p53,

p21Cip1, and p19Arf, which converge to inhibit cyclin-dependent kinases (CDKs) and retinoblastoma protein

(Rb)25-27.

An important distinguishing characteristic of senescence programs is the SASP. Developmental senescence is also distinct in this respect as shown by transcriptome analyses revealing only a subset of common gene expression changes to other senescence programs9. The SASP composition during

regeneration and repair in vivo, for example during cutaneous wound healing is marked by production of Ccn1 and PDGF-AA which play a central role in the induction and maintenance of the senescent state11, 28.

Immune surveillance of oncogene-induced senescent (OIS) cells in the liver is promoted by the monocyte chemo-attractant Mcp1 (or Ccl2)16, 17. Conversely, chronic senescent cells acquired during ageing or

therapy display a high degree of variability between stressor, tissue type, and species30, but consistently

induce IL-6 and Pai-1 in vivo20. Based on these studies, IL-6, IL-1α, Pai-1, and Mcp1 seem to be canonical

SASP factors induced under most circumstances. Developmental senescence

In recent years, several studies have implicated senescence in embryogenesis and tissue remodeling9, 10, 31 (Figure 2 & Table 1). Senescent cells transiently appear in several embryonic structures,

including the mesonephros, the endolymphatic sac of the inner ear, and the apical ectodermal ridge of the limbs9, 10. Developmental senescence seemingly is a mechanism for fine-tuning organogenesis and is

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Chapter 2

developmental senescence, develop successfully by activating compensatory mechanisms such as apoptosis9. In the mesonephros, senescent cells appear in the mesonephric tubules at murine embryonic

day 12.5-14.5 and in approximately 9-week-old human embryos9. Senescent cell accumulation precedes

macrophage infiltration and is thought to attract immune cells facilitating mesonephros regression and ultimately clearance of senescent cells upon completion9, 10. Loss of developmental senescence in p21Cip1

knockout mice shows compensatory caspase activation, apoptosis, and delayed, but ultimately successful, mesonephros regression9. In the female Wolffian duct, however, p21Cip1 knockout increases the incidence

of vaginal septa in females and results in fewer offspring9. Collectively, these studies indicate that

senescence functions as a complementary mechanism to apoptosis for the elimination of specific groups of cells in the development of certain tissues9, 35. However, why senescence would be favored over

apoptosis in certain structures such as the mesonephros is unclear. Senescence in regeneration

Acute regenerative senescence is usually induced after an initial insult as part of a healing or repair response (Figure 2 & Table 1). For example, in the early stages of cutaneous wound healing, senescent cells induce myofibroblast differentiation and promote wound closure through paracrine signaling11.

Senescent cell depletion via targeted killing of p16Ink4a-positive cells during wound healing in mice impairs Box 1: Common markers of cellular senescence

In vivo identification of senescent cells is challenging, especially considering their diversity and heterogeneity. However, there seems consensus in the field that some senescence phenotypes are considered necessary senescence markers. The most used marker is staining for senescence-associated β-galactosidase (SA-β-Gal) activity at pH=6.0, which is a reflection of increased lysosomal content of senescent cells1. Alternatively, staining for the lysosomal structure lipofuscin may be used

under some circumstances2. A key senescence characteristic is the absence of proliferation signals

(e.g. Ki-67 or Mcm2 absence, lack of bromodeoxyuridine, BrdU or ethynyldeoxyuridine, EdU incorporation) and increase in cyclin-dependent kinase (CDK) inhibitors, Cdkn1a (p21Cip1), Cdkn2a

(encoding the proteins p16Ink4a andp19Arf (in mouse) p14Arf (in human)), Cdkn1b (p27Kip1) or Cdkn2b

(p15Ink4b). Other important features of many senescence mechanisms are a DNA damage response

(e.g. detection via γ-H2A.X foci or 53BP1 foci)3 and senescence‐associated heterochromatic foci, SAHF

(marked by HP1-γ, H3K9me3 or macroH2A)6. One of the most physiological relevant but often

under-studied feature of senescent cells is the SASP. For our basic understanding and for developing targeted senescence therapies, it is essential to describe the composition of the SASP quantitatively and qualitatively. Canonical SASP factors such as IL-6, IL-1α, Pai-1, Mcp1/Ccl2, (see also Table 1) as well as other patho-physiologically relevant SASP markers should be assessed routinely.

Importantly, neither of these markers is specific for senescent cells and, therefore, a panel of markers must be used to demonstrate presence of senescence. For example, SA-β-Gal activity can be detected in osteoclasts8, macrophage subpopulations21 or due to increased cell confluence in vitro24; p19Arf and

p16Ink4a are expressed in immune cells29 or in tumors with Rb inactivation32. Further proof of senescence

and help to identify the cell type undergoing senescence in tissues can be achieved by using reporter animal models such as the mouse models for p16Ink4a-mediated senescence p16-LUC33, 34, p16-3MR11

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Figure 1: Simplified model of the core senescence-inducing signaling pathways. Developmental senescence is thought to be induced as part of a physiological program that is dependent on p21Cip1, but not on other cyclin-dependent kinase (CDK)

inhibitors such as p19Arf, p16Ink4a or p539, 10. On the other hand, stress- and insult- induced senescence types, regenerative

senescence, cancer-associated senescence and senescence in ageing, diseases and therapy, can vary substantially dependent on the stimulus, context or cell type. Stress-induced senescence mechanisms usually constitute of activation of a signaling cascade involving a DNA damage response (DDR) via Atm or Atr kinases, p53 activation and increased p21Cip1 transcription36, 37 and/or

induction of p16Ink4a via multiple signaling pathways38. Notably, murine oncogene-induced senescence relies more on p19Arf,

whereas human cells are more dependent on p16Ink4a signaling39, 40. Both, p21Cip1 and p16Ink4a activation result in inhibition of CDK

complexes and prevention of Retinoblastoma protein (Rb) phosphorylation25, 26.

the kinetics of the healing process, but does not impact the total time to full closure11, 20, suggesting that

senescence in the context of tissue regeneration has a dispensable, fine-tuning role. Interestingly, in the later stages of wound healing28 and after cardiac infarction41, senescent fibroblasts limit tissue fibrosis and

further tissue damage by secreting anti-fibrotic factors, such as matrix metalloproteinases (MMPs). However, prolonged, chronic senescent cell signaling and impaired elimination, can lead to detrimental consequences. Upon liver damage in mice for example, senescent hepatic stellate cells reduce fibrosis42,

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Chapter 2

Renal tubule and interstitial cells become senescent after unilateral ureteral obstruction45. Preventing the

formation of senescence through p16Ink4a inactivation increases renal fibrosis, indicating that senescence

in this context is part of an anti-fibrotic mechanism45.Acute regenerative senescence is also beneficial

immediately after ischemic reperfusion injury (IRI) in murine kidneys. Within two days after IRI, p16Ink4a and

p21Cip1 are induced in tubule cells46 and p21Cip1 knockout mice exhibit impaired renal recovery, higher renal

damage, and mortality after IRI47. Additionally, mice with proximal tubules lacking Atg5, a protein involved

in autophagy and essential for senescence induction48 and SASP production49, impairs renal senescence46

causing increased renal damage and cell death in the acute phase after IRI46, 50, 51. The dual role of

senescent cells after IRI is illustrated in p16Ink4a knockoutmice, where detrimental long-term consequences

after IRI, such as interstitial fibrosis, tubular atrophy, and impaired kidney function are ameliorated52. These

examples suggest time-dependent contributions of senescent cells to renal injury, with early positive effects, followed by detrimental long-term consequences. It will now be important to understand how deleterious senescent cells form, evade elimination by the immune system, and exert their tissue-deteriorating properties.

Dual role of senescence in cancer

Senescence is a potent tumor-suppressive mechanism, preventing expansion of damaged and pre-neoplastic cells (Figure 2) with hyper-activation of oncogenes or deactivation of tumor-suppressor proteins engaging the senescence program40, 53. Conditional inactivation of the tumor-suppressor Apc in murine

renal epithelial cells induces senescence, a response that inhibits formation of renal carcinomas as demonstrated by the co-inactivation of p21Cip1 or p16Ink4a/p19Arf and Apc resulting in an earlier onset of renal

carcinoma54. Loss of Vhl, a key renal tumor suppressor gene, also induces senescence in the renal

epithelium55, underscoring the importance of senescence as a cancer-protective mechanism in the kidney.

On the other hand malignant cancers exploit the secretome of senescent stromal cells to advance growth, angiogenesis, epithelial-to-mesenchymal transition (EMT), immune cell evasion, and metastasis15, 56, 57.

Strikingly, tumor cells themselves can promote stromal cell senescence via paracrine signaling57, 58, for

example, Gro-1 (Cxcl1) has been shown to induce senescence of stromal fibroblasts in vitro58. Identification

of senescent cells in human and mouse tumors is consistent with a cancer-promoting role of senescent cells33, 57, 58. Removal of p16Ink4a-positive cells using the INK-ATTAC mouse model delayed tumor latency

without impacting tumor incidence or spectrum20, indicating that naturally occurring senescent cells

stimulate tumor progression. Whether this also applies to renal cancers remains to be experimentally tested.

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Figure 2: Diverse roles of senescent cells during the life of the kidney. For references see text. Stress-induced chronic senescence

Acute senescence in development, regeneration, and oncogene-activation is viewed as beneficial, while chronic senescence due to gradually increasing macromolecular damage and other chronic stresses (cellular “wear and tear”) commonly observed during ageing and disease, is thought to be largely detrimental (Figure 2 & Table 1). Discriminating whether senescence in a given context is beneficial or detrimental requires the use of model systems that allow for intervention during the acquisition of senescent cells to determine their relative importance. The reason and kinetics for senescent cell accumulation during ageing and disease are incompletely understood. Age-related senescent cell accumulation coincides with reduced immune system function59, 60 implying that impaired immune function allows senescent cells to

evade clearance. Age-related senescent cell accumulation in mice has been shown to shorten lifespan, promote tissue deterioration, and impair the function of several organs, including the heart, vasculature, adipose tissue, and kidney20. It is thought that the pro-inflammatory properties of the SASP61 and the cell

autonomous effects of reduced regenerative capacity of stem cells62-65 are contributing factors. Stresses or

damage from disease may also induce senescence. However, whether senescence initiates or promotes disease progression is unclear. For example, high-fat diet promotes obesity, type 2 diabetes, and senescence in adipose tissue66, 67, pancreatic β-cell islets68, and in arteries during arteriosclerosis69, 70.

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Chapter 2

Furthermore, mechanical stress caused by hypertension can induce senescence in circulating endothelial progenitor cells71, 72, as well as in heart and kidney73. Exogenous stresses from therapeutic intervention,

such as organ transplant74, 75 or chemotherapy with cytotoxic drugs76, 77 or irradiation78, 79 also stimulate

senescence. The kidney encounters stress or damage from many sources. Below, we discuss stress-induced senescence in the kidney in various contexts, including renal ageing, disease, and therapy-stress-induced damage (Table 1).

Senescence in renal ageing

The kidney undergoes many structural and functional changes with ageing including glomerulosclerosis, tubular atrophy, interstitial fibrosis, arteriosclerosis, loss and hypertrophy of nephrons, and tubular diverticula80-83 (Figure 3a & Table 1). Macroscopic age-related changes include reduced cortical volume,

Table 1. Senescent cells in renal aging, disease, and therapy-induced damage. EM- electron microscopy, IHC- immunohistochemistry, W- western blotting, IF- immunofluorescence

Renal Defect Model Evidence (method) Cell Type(s) Potential Impact Reference(s)

Age-Related

Glomerulosclerosis mouse SA-β-Gal (EM)/p16 Ink4a,

p19Arf, p21Cip1 (qPCR) Proximal tubules Detrimental Baker et al. 2016 human p16INK4A, p53 (IHC) cortex- tubules, glomeruli, interstitium, and arteries Detrimental Melk et al. 2004 Interstitial fibrosis mouse and rat

SA-β-Gal, p16Ink4a (IHC/qPCR), p19Arf

(qPCR) Cortical tubules and glomeruli Detrimental Krishnamurthy et al. 2004

human p16

INK4A, p53, TGFβ1

(IHC) Cortex – tubules, glomeruli interstitium, arteries Detrimental Melk et al. 2004 Nephron atrophy human p16INK4A, p27KIP1 (IHC) Cortical tubules and interstitium Detrimental Chkhotua et al. 2005

human p16

INK4A, p53, p14ARF,

TGFβ1 (IHC) Cortex – tubules glomeruli, interstitium and arteries Detrimental Melk et al. 2004

Disease Associated

Acute kidney injury

(AKI) mouse SA-β-Gal, p21Cip1 (W) Tubules Detrimental Clements et al. 2013

mouse SA-β-Gal/γ-H2A.X (IF)/ p16Ink4a, p19Arf (qPCR) Tubules Detrimental Baisantry et al. 2016

mouse p21

Cip1 (IHC)/p16Ink4a,

p21Cip1 (IHC) Tubules Beneficial/Detrimental Megyesi et al. 2001/ Hochegger et al. 2006 IgA nephropathy human SA-β-Gal, p16

INK4A,

p21CIP1 (IHC) Tubules Detrimental Liu et al. 2012 Diabetic nephropathy human SA-β-Gal, p16INK4A (IHC) Glomeruli, and tubules Detrimental Verzola et al. 2008

mouse SA-β-Gal, p21Cip1 (IHC) Glomeruli, and tubules Detrimental Kitada et al. 2014 Membranous glomerulopathy (MGP), focal segmental glomerular sclerosis (FSGS), and minimal change disease (MCD) human p16

INK4A, p21CIP1 (IHC

both, p21- FSGS only) Glomeruli, tubules, and interstitium Detrimental Sis et al. 2007 Autosomal dominant

polycystic kidney

disease (ADPKD) human and rat p21CIP1/Cip1 (IHC, W) N/A- reduced; induction in tubules ameliorated disease Beneficial Park et al. 2007 Nephronophthisis

(NPHP) mouse SA-β-Gal, p16Ink4a (W) Tubules Beneficial/Detrimental? Lu et al. 2016

Chronic kidney

disease (CKD) cat SA-β-Gal Proximal and distal tubules Detrimental Quimby et al. 2013

Therapy-Induced

Cisplatin mouse p21Cip1 , p27Kip1 (IHC) Outer medulla Detrimental? Zhou et al. 2004

Renal transplant human SA-β-Gal, p16INK4A (IHC) Glomeruli, tubules, and interstitium Detrimental Melk et al. 2005

Table 1: Senescent cells in renal ageing, disease, and therapy-induced damage. EM- electron microscopy, IHC- immunohistochemistry, W- western blotting, IF- immunofluorescence.

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renal cysts and tumors, atherosclerosis of renal arteries, parenchymal calcifications and cortical scars84-86.

Reductions in cortical volume are largely due to glomerulosclerosis and tubule atrophy, whereas parenchymal volume is preserved due to compensatory tubular hypertrophy87. At the functional level, the

glomerular filtration rate (GFR), the ability to conserve and secrete sodium, and the urine concentrating and diluting abilities have all been reported to decline with ageing88, 89. Although GFRs gradually decrease over

time in most individuals, renal function typically remains within what is considered a normal range90 and

does not predict end-stage renal disease (ESRD)87.

The regenerative potential upon acute kidney injury (AKI) or kidney transplantation, decreases with ageing in both mice and humans91-94. Furthermore, kidneys from old donors have reduced function95 and

transplantation success96, 97, poor regeneration after acute rejection, and higher rates of graft functional

loss due to chronic degeneration98, 99. Reduced GFR and nephron numbers in aged kidneys may also cause

toxic accumulation of renally-cleared medications in aged individuals87. Given that the kidney is an organ

with a relatively large functional reserve capacity, it is hard to assess when age-related changes transition into kidney disease. Regardless, as for most organs, it is clear that kidney ageing is a major risk factor for disease.

Senescent cells accumulate with age in the kidney and correlate with functional decline and features of age-related deterioration (Figure 3a & Table 1). Recent studies in which naturally occurring p16Ink4a-expressing cells were cleared from mice from one year on provided compelling evidence that

senescent cells contribute to ageing, including aspects of kidney ageing20. Specifically, using INK-ATTAC

transgenic mice, p16Ink4a-expressing senescent cells were systemically removed beginning at midlife, when

the number of senescent cells is relatively low, and continued with treatment throughout their life. Clearance markedly extended lifespan irrespective of gender and genetic background. Mice from which senescent cells were removed showed attenuated glomerulosclerosis and retained youthful blood urea nitrogen (BUN) levels with ageing, indicating that the senescence program actively contributes to these age-related pathological alterations. Senescence occurred in proximal tubule cells, elevating angiotension receptor 1a (Agtr1a) expression throughout the kidney, thus hyperactivating the local renin-angiotensin-aldosterone system (RAAS) and causing glomerulosclerosis.

Others have observed cells with senescent features in the cortical tubules, glomeruli, and interstitium of aged mouse20, 100 and human101, 102 kidneys. Nephron atrophy seemingly is a downstream

consequence of glomerulosclerosis, and may therefore also be indirectly linked to cellular senescence. However, it may also be driven by senescence of tubule cells. The effect of senescent cells on interstitial fibrosis is complicated. In some instances, senescence appears to inhibit fibrosis45, whereas other studies

have shown positive correlations between senescent cell accumulation and fibrosis in the kidney during ageing102-105 and disease52, 73, 75, 92, 106, 107, which may potentially be explained by the dual action of

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Chapter 2

Although, induction of senescence following renal transplant may promote wound closure and limit tissue fibrosis, the presence of chronic senescent cells in graft tissue is detrimental. Therefore, the combined use of chronological age and renal p16Ink4a levels may provide an excellent prognosticator of renal allograft

function post-transplantationally95, 108. Indeed, kidneys transplanted from old donor mice, exhibited

increased susceptibility to transplant stress and enhanced induction of p16Ink4a, compared to young

a

b

c

Renal aging Renal disease Therapy-induced damage Glomerulosclerosis Interstitial Fibrosis Nephron Atrophy CKD MGP, FSGS, & MCD IgA Nephropathy Diabetic Nephropathy NPHP AKI Renal Transplant Interstitial fibroblast Senescent cell SASP

Figure 3: Age-, pathology-, and therapy-related defects in the kidney are associated with senescent cell accumulation. Senescent cells have been characterized in a variety of locations within the nephron in association with renal ageing (a), disease (b), and therapy-induced damage (c), and are depicted here. In each instance, the renal defect and senescent cell location are associated by color. Senescent cells are present in aged kidneys (a) in associated with glomerulosclerosis (red, proximal tubules), interstitial fibrosis (purple, proximal and collecting tubules), and nephron atrophy (blue, age-related senescence cells have been described in the glomeruli, tubules, and interstitial cells). p16Ink4a-positive or senescent cells are also present in kidneys in

association with several renal diseases (b), including: chronic kidney disease (CKD, red, proximal and distal tubules); membraneous glomerulopathy (MGP), focal segmental glomerular sclerosis (FSGS), and minimal change disease (MCD, orange, glomeruli, tubules, and interstitial cells); immunoglobulin A (IgA) nephropathy (purple, tubules); diabetic nephropathy (blue, glomeruli and tubules); nephronophthisis (NPHP, green, tubules); and acute kidney injury (AKI, pink, tubules). Senescent cells are also induced due to transplant stresses (c) (purple, glomeruli, tubules, and interstitial cells).

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donors109. Senescent cells may negatively impact the initial phase after transplantation, where epithelial

cell proliferation is required110, and grafts from old donor mice exhibit reduced proliferation of renal tubule

cells post-transplantation compared to young mice109.

Renal pathologies and senescence

Senescent cells seem to accumulate in several renal diseases as will be reviewed below (Figure 3b & Table 1). Given the emerging evidence that senescent cells are causally implicated in age-related renal dysfunction, further study into the role of senescence in kidney diseases is likely to improve our understanding of the molecular mechanism underlying kidney pathologies and the potential usefulness of developing new therapies that target senescent cells, referred to as senotherapies111.

Acute kidney injury

Acute kidney injury (AKI) is defined as an abrupt loss or disruption of kidney function. Although it typically occurs as a secondary consequence, it is a primary driver of renal damage and chronic kidney disease (CKD) in humans112. One of the major risk factors of AKI is age, but other factors include infections,

glomerulonephritis, sepsis, and toxic insults, such as contrast agents used for imaging or therapeutic agents113-115. The impact of maladaptive repair following AKI can be highly detrimental in the elderly.

Consistent with this, renal function and mortality rates of rodents after IRI are markedly aggravated with ageing, and associated with increased interstitial fibrosis, oxidative stress, inflammation, p53 and p21Cip1

levels, SA-β-Gal positive tubules, and reduced proliferation92, 116. These results suggest that increased

levels of basal chronic senescence and reduced regenerative potential of aged kidneys have dramatic consequences on recovery after injury. It is possible that senescence in tubules prevents proliferation that is required for repair of damaged cells or aggravates the development of fibrosis.

Glomerulonephritis

Immunoglobulin A (IgA) nephropathy, a form of glomerulonephritis, is an autoimmune disease acquired independently of age due to genetic or environmental features, where IgA antibody deposits cause inflammation and damage to the glomerulus, leading to proteinuria or hematuria117. Disease progression

correlates with telomere shortening, a causative feature of senescence and premature ageing118.

Consistent with this, increased expression of p21Cip1 and p16Ink4a, along with increased SA-β-Gal activity

were detected in renal tubules from patient biopsies106. These features correlated with disease progression,

including increased fibrosis and hypertension, and reduced renal function106, suggesting a potentially

important role of cellular senescence in the progression of IgA nephropathy, however, whether this is simply associated with tissue damage and not contributing to disease pathology remains to be determined. Diabetes and diabetic nephropathy

Aberrant glucose metabolism, as observed in diabetes, is associated with serious long-term cardiac, vascular, and renal complications and various features of ageing, including sarcopenia, functional disability,

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Chapter 2

frailty, and early mortality in older adults119. In addition, diabetes has been associated with cellular

senescence in pancreatic β cells68 and adipose tissue66, 67. In the kidney, impaired glucose metabolism can

lead to the development of diabetic nephropathy, a progressive kidney disease characterized by glomerular scarring, reduced kidney function, and proteinuria120. Diabetic nephropathy is the most common cause of

CKD and ESRD, and is associated with an increased risk of cardiovascular disease121.

Tubule cells, podocytes, mesangial-endothelial, and vascular-endothelial cells become p16Ink4a-

and SA-β-Gal-positive in type 2 diabetic nephropathy patients107 and a streptozotocin-induced mouse

model for type 1 diabetes122. Furthermore, several groups have shown a direct link between hyperglycemia

and the induction of senescence in vitro in mesangial123-125 and proximal tubule cells122, and in vivo by

normalization of glucose levels in a mouse model of type 1 diabetes122. In addition, p21Cip1 and p27Kip1

depletion in diabetic nephropathy models showed reduced proteinuria, glomerular hypertrophy, and tubulointerstitial damage126, 127. Together, this suggests that cellular senescence plays a role in the

pathogenesis of diabetic nephropathy, and that hyperglycemia is an important driver of this phenotype. The systemic impact of diabetic nephropathy has also been associated with cellular senescence, as premature senescence was observed in skin fibroblasts from patients with insulin-dependent diabetic nephropathy128,

suggesting a potential relation between renal disease and premature ageing. Nephrotic syndrome

A number of other glomerular diseases that cause nephrotic syndrome have also been associated with increased levels of p16Ink4a. Evaluation of renal biopsies from membranous glomerulopathy (MGP), focal

segmental glomerular sclerosis (FSGS), and minimal change disease (MCD) patients showed a dramatic increase in p16Ink4a levels in glomeruli, interstitium, and tubules, independent of age. This correlated with

the degree of interstitial fibrosis and tubular atrophy105. In patients with tubulointerstitial fibrosis,

inflammation was associated with increased p16Ink4a in interstitial and tubule cells105. Although, elevated

p16Ink4a alone is not sufficient to conclude that cells are senescent, increased p21Cip1 levels were also

detected in biopsies from patients with FSGS105. It will be important to determine if additional markers of

senescence are present in these diseased tissues, and how senescent cells may contribute to disease initiation or progression.

Polycystic kidney disease

Polycystic kidney disease (PKD) is characterized by the formation of fluid filled cysts in the renal tubules that progressively expand, resulting in damage to the remaining functional tissue and ultimately leading to ESRD129. The most common form of PKD is autosomal dominant polycystic kidney disease (ADPKD), which

leads to ESRD in the fifth to seventh decade of life129. In general, PKDs are characterized as ciliopathies,

as they arise from mutations in genes encoding primary cilia-related proteins and are thought to primarily impact the ciliated epithelial cells lining the renal tubules130. PKD is also characterized by increased

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ADPKD patients and a rat model of PKD133. Treatment with the CDK inhibitor roscovitine restored p21Cip1

levels in vitro and in vivo, increased SA-β-Gal staining in vitro, decreased renal tubule cell proliferation, and ameliorated disease progression in an ADPKD mouse model133-135.

In contrast, cellular senescence is induced in nephronophthisis (NPHP), a syndromic form of PKD, and the most common cause of renal failure in the first three decades of life136. NPHP is characterized by

the formation of renal cysts, but unlike the massive expansion in renal volume typically observed in ADPKD, NPHP is associated with a progressive reduction in renal volume due to tubular atrophy and interstitial fibrosis136. A mouse model of NPHP shows reduced Ki-67-positive renal cells, increased levels of p16Ink4a,

and an increase in SA-β-Gal-positive renal tubules137. When these mice were crossed onto a

non-orthologous model of PKD (conditional Kif3a knockout), the massive cystic expansion of the Kif3a model was dramatically reduced, in combination with reduced proliferation, increased DNA damage, and an increase in SA-β-Gal-positive tubules137.

Together, this suggests that cellular senescence may be important in multiple forms of PKD, and as discussed previously, for various biological processes. Although, senescence seems to play a beneficial role in some forms of PKD, such as ADPKD, whether the role of senescence in NPHP is beneficial or detrimental requires further investigation. It is interesting, however, that an association between increased senescence and fibrosis can be observed in this disease model, suggesting that finding a balance between mild cystic expansion and fibrosis may be important for amelioration of these diseases.

Chronic kidney disease

CKD is defined as a gradual decline in kidney function over time, and can be caused by all of the pathologies described above, but is primarily a consequence of diabetes or diabetic nephropathy113, 114, 138. Individuals

with CKD are at risk of developing other age-related renal pathologies, including glomerulosclerosis139 and

AKI113, and young patients with CKD frequently exhibit features of early ageing, including vascular ageing,

muscle wasting, bone disease, cognitive dysfunction, and frailty, highlighting the importance of proper renal function and the systemic impacts of kidney function impairment140, 141.

Features of cellular senescence have been associated with CKD in various animal models, including a murine model of adenine nephropathy, where an increase in inflammatory markers of the SASP were observed142, and a feline model of CKD, where telomere shortening and increased SA-β-Gal were

detected in proximal and distal collecting tubules of CKD cats compared to both young and aged cats143.

Correlations between the systemic consequences of CKD and cellular senescence are also apparent. Bone marrow mesenchymal stem cells from rats with CKD exhibited premature senescence144, increased

transcription of p53 and Rb, and reduced proliferation were observed in lymphocytes from CKD patients144.

Although these correlations suggest a relationship between cellular senescence and CKD, additional studies are required to determine the role of senescence in disease progression and whether it is a cause or consequence of pathogenesis.

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Chapter 2 Senescence as a consequence of therapy

Cancer therapy-induced collateral damage to normal tissue represents a serious health concern for patients, especially survivors of childhood cancers, who have a high risk for developing chronic pathologies, including secondary cancers, cardiovascular disease, renal dysfunction, musculoskeletal problems, and endocrinopathies145. For instance, cisplatin causes renal damage and induces a marked increase in p21Cip1

in kidney tissue of rodents (Figure 3c & Table 1)146, 147. Furthermore, the incidence of cisplatin nephrotoxicity

increases with age in both mice and humans148. Although these findings are suggestive of a relationship

between chemotherapy and senescence-induction in kidney, formal evidence is currently lacking.

Perhaps the most well described therapy-induced acquisition of renal senescence occurs with transplantation. Cellular senescence in advanced age organ donors reduces transplant success and organ longevity95, 108. In addition, stresses during and after the transplantation process such as IRI, acute rejection,

or hypertension may also induce senescence91. Indeed, renal transplantation in rats promotes substantial

telomere shortening, and increased p21Cip1 and p16Ink4a expression in graft tissue74. Likewise, human

post-transplantation biopsies exhibit high renal p16Ink4a levels in tubules, tubular atrophy, interstitial fibrosis, and

high numbers of, glomerular, and interstitial cells (Figure 3c & Table 1)75, 105. Furthermore, mice receiving

kidney transplants from Cdkn2a null donors had markedly reduced renal damage and significantly better survival, which correlated with increased tubule cell proliferation, and significantly fewer senescent cells, defined as Ki-67-negative, γ-H2A.X-positive cells52.

Allograft rejection is also associated with cellular senescence, and glomerular, tubule, and interstitial cells from transplant patient rejected grafts express elevated levels of p16Ink4a and p27Kip1, which correlated

with chronic allograft nephropathy grade101. In addition, chronically rejected kidneys from a rat model of

chronic rejection also exhibited increased p16Ink4a and p21Cip1 expression, and increased SA-β-Gal activity74.

Based on current knowledge, cellular senescence seems to have merely negative effects at all stages of kidney transplantation and is ultimately associated with allograft rejection and chronic allograft nephropathy. Senotherapy

As drivers of ageing and disease in various tissues and organs, including kidney, senescent cells have emerged as promising new targets for a therapeutic intervention known as senotherapy (Figure 4)111.

However, given the complex roles of senescence in different biological processes and tissues, proper timing and delivery of senotherapy has to be carefully considered. Targeted drug delivery to kidneys provides an exciting opportunity for senotherapy in this organ. Such systems include polyvinylpyrrolidone (PVP)-derivates149, low-molecular-weight-protein (LMWP)-carriers150, ligand-conjugates specific for

renal-associated receptors151, and a recently-developed targeting peptide (KKEEE)3K152. These approaches

target tubule cells, a renal cell type that displays features of senescence with ageing and pathology. Targeting other renal cell types, such as mesangial cells, which become senescent as a consequence of hypertension153-155 and diabetic nephropathy107, is more challenging, but could be accomplished using

nanoparticles156 or liposomes157. Another promising targeting approach, used in IRI, is intravenous

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Figure 4: Senotherapeutic targeting strategies. Pro-senescent interventions act on senescence-inducing pathways e.g. Cdk4/6 inhibition. Anti-senescence strategies include prevention, cell lysis and SASP modulation. Senescence prevention is achieved by mitigating the pro-senescent stressors. Several drugs and life-style interventions currently considered life extending, such as caloric restriction, fall into this category. Senescent cells can be removed by senolysis by interfering with the anti-apoptotic and pro-survival signaling. Navitoclax, an inhibitor of Bcl-2, Bcl-xL and Bcl-w, has been shown to selectively induce apoptosis in senescent cells in vitro and in vivo. Besides removal, the modulation of SASP is another option how to interfere with senescent cells. This strategy includes inhibition of pro-inflammatory pathways or gene expression of SASP factors via Brd4 inhibition. For references see text.

Senescence prevention

Potential interventions protecting cells against macromolecular damage may include healthy diet, exercise and avoidance of lifestyle-related stresses such as cigarette smoking, but may also include drugs that have been shown to extend healthy lifespan in rodents, metformin and rapamycin. Restricted calorie intake extends health- and lifespan of rodents, and reduces oxidative stress and the age-related rise in circulating pro-inflammatory factors, thereby mitigating known inducers of senescence159, 160. In addition, kidneys from

aged calorie restricted rats have reduced p16Ink4a levels, SA-β-Gal positivity, glomerular volume, and

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Chapter 2

important regulator of energy metabolism that involves AMP-activated protein kinase (AMPK). Several drugs targeting this pathway are available including compounds regarded as lifespan-extending, such as the mTOR inhibitor, rapamycin, or the AMPK activator, metformin. Rapamycin protects against mTOR-induced cellular senescence162, 163 and phosphate induced premature ageing164, and metformin prevents

the increase of p16Ink4a, p21Cip1, and inflammatory SASP-related cytokines165 in irradiation-induced cells.

Metformin also reduces ROS production in cultured podocytes166, prevents diabetes-induced renal

hypertrophy167, protects the kidney from gentamicin- and cisplatin-induced renal damage in mice168,169,

suggesting a possible positive role in reducing kidney senescence burden. The main challenge when developing interventions that prevent senescence is to ensure that stressed cells at risk of neoplastic transformation are fully capable of activating the senescence program.

Senescent cell clearance

From the perspective of cancer, elimination of cells after they have become senescent is perhaps the safest senotherapeutic approach. As studies in mice suggest14, 20, agents capable of removing senescent cells,

termed senolytics, may soon become tools to treat age-related disease and promote healthy ageing. Senolysis can perhaps be best achieved by following the blueprint for killing cancer cells, including activation of the immune system, inhibition of pro-survival pathways or activation of pro-apoptotic pathways. In the coming years, it will be imperative to describe differences in properties relevant for the survival of senescent cells based on the tissue of origin and mechanism of senescence induction, which may greatly affect senolytic efficacy. Through these efforts, cell type, tissue or context specific senotherapies may show promise in reducing potential off-target effects. Furthermore, identification of non-senescent cells relying on pathways targeted by senotherapies will facilitate understanding the side effects of treatment.

It is assumed that in many organs, senescent cells are targeted by the immune system, although this has only been clearly demonstrated in the liver170. In the context of developmental, regenerative and

cancer-protective senescence, these processes appear to clear the majority of senescent cells. Understanding how chronic senescent cells resist clearance may be informative for developing immune-based therapies. Although the mechanisms mediating immune surveillance vary in different settings, the innate immune response appears to be particularly important. For example, impairing NK-cell-mediated clearance of activated stellate cells in a liver fibrosis model, leads to increased liver fibrosis in mice44. Furthermore, the

SASP of senescent liver cells activates macrophages and induces their polarization towards the secretory M1 type, leading to senescent cell clearance19. Boosting these processes could be an important tool in

anti-senescence therapies.

ABT-263 (Navitoclax), an inhibitor of pro-survival proteins Bcl-2, Bcl-xL (Bcl2l1), and Bcl-w (Bcl2l2), has been identified as a first generation senolytic171. Multiple laboratories have independently demonstrated

its senolytic activity in the context of irradiation, replicative, and oncogene-induced senescence. However, whether other senescence programs are also targeted has not been investigated yet. Interestingly, Bcl-xL is consistently increased in DNA damage, replication stress, and oncogene-induced senescence172, but is

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down-regulated in developmental senescence of the mesonephros9, indicating that different programs of

senescence have a different involvement of Bcl-xL. Combinatorial treatment with quercetin, a flavonol with antioxidant properties that also inhibits a broad spectrum of protein kinases173, and dasatinib, an inhibitor

of several tyrosine kinases174, has also been used to target senescent cells175. However, it will be important

to confirm that effects of these and other senolytic compounds are due to senescent cell removal, as the current strategies target pathways that are not unique to senescent cells.

Removal of renal senescent cells has the potential to be beneficial in ageing and in many kidney diseases, but this has not been experimentally confirmed. Feasibility will need to be assessed for each kidney condition separately. Kidney transplantation is one instance where senolysis could be highly relevant. Pre-treating donors well before explanting the kidney or perfusion of the organ after removal with senolytics may reduce the chronic senescent cell burden and lower the risk of transplant rejection. Importantly, during the healing period after implantation, senescent cells promote cutaneous wound healing11; therefore, anti-senescence strategies may initially need to be avoided. However, senescent cells

from post-operative stresses or accumulating due to immuno-suppressant therapies could be removed by senolysis after sufficient healing has occurred.

SASP modulation

The SASP is a key feature of cellular senescence that has profound effects on neighboring cells. Interference with the SASP can be achieved by inhibiting pro-inflammatory signaling pathways, such as NF-κB or p38-MAPK signaling176, 177, however, these interventions will affect non-senescent cells too178.

Interestingly, rapamycin and metformin also inhibit the SASP in vitro through mTOR, p38-MAPK, MK2 (Mapkapk2) or NF-κB signaling. Brd4 inhibitors seem to modulate the SASP with high specificity179. Brd4

is a protein that binds to acetylated lysines on histones, thereby marking open chromatin regions including active enhancer elements that regulate components of the SASP179. Brd4 inhibitors allowed senescent cells

to escape immune surveillance in a mouse model of oncogene-induced senescence179. SASP modulation

would not only reduce the chronic burden of senescent cell-associated inflammation, but also impair immune-mediated clearance, potentially leading to excessive accumulation of senescent cells. Thus, the implication of these interventions on the progression of senescent cell accumulation needs to be further studied. It is also not clear whether the aforementioned interventions attenuate accumulation of all senescence programs in a similar fashion, or more likely, impact senescence of specific cell types induced by a particular stressor.

Pro-senescence strategies

Induction of senescence in cancer cells is recognized as a favorable outcome to limit tumor progression180, 181, and is induced by cytotoxic and radiation therapies182, which has led to the development of compounds

inducing senescence of cancer cells. Several Cdk4/6 inhibitors have been developed for this purpose, including abemaciclib, palbociclib and ribociclib, all three of which are being tested for therapeutic efficacy

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Chapter 2

against various cancer types183. Palbociclib has also been used in a model of AKI, where treatment after

IRI protected against DNA damage, apoptosis, and subsequent kidney damage184. An obvious concern of

the use of senescence-inducing drugs to combat cancer would be the potential for long-term detrimental effects due to the accumulation of excessive amounts of senescent cells. Perhaps such negative side effects can easily be avoided by combining pro-senescence therapy with senolysis.

Conclusions

Developmental, regenerative, cancer-related, and age- and disease-related senescent cells exhibit considerable variability in their effects, mechanisms of induction, secreting factors, and lifetime. If viewed in the “short-term”, senescence contributes in a beneficial way to several physiological and pathological processes. These transient benefits are seemingly outweighed by the detrimental consequences of “long-term” senescence if there is inefficient clearance and accumulation of these cells. Unfortunately, there are currently no distinguishing characteristics to discriminate between these possibilities, which requires further investigation and the use of model systems that allow for targeting of senescent cells. Using INK-ATTAC transgenic mice, senescent cell removal extended healthy lifespan, including improved kidney function. No detrimental effects of long-term administration to eliminate senescent cells were observed, indicating that senescent cell accumulation during normal ageing is primarily detrimental. The beneficial impacts of senescent cells may turn out to be modulatory in nature and therefore manageable once we understand what senescent cells are truly contributing during various physiological processes. Tubule cells are frequently affected in the nephron in ageing and disease, therefore it is necessary to focus on detailed characterization of these cells in healthy and diseased patients. Additionally, further characterization of the senescent properties of all renal cell types in ageing and pathology is warranted, as hints of senescence have been alluded to. A limitation of these studies is that most have only used a subset of established senescence markers, which highlights the need for further investigation. Although the contribution of senescent cells to renal ageing and pathology is only beginning to be elucidated, the development of therapeutic tools to support healthy kidney ageing, ameliorate kidney disease, and improve the success of renal transplantation will likely involve modulation of the senescence program in this vital organ.

Author contributions

All authors researched the data, discussed the article’s content, wrote the text and reviewed or edited the article before submission.

Competing interests statement

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