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Home , Cellular senescence

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Cellular in the Kidney

Marie-Helena Docherty,1 Eoin D. O’Sullivan,1,2 Joseph V. Bonventre,3 and David A. Ferenbach1,2

1Department of Renal Medicine, Royal Infirmary of Edinburgh, Edinburgh, UK; 2Centre for Inflammation Research, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK; and 3Renal Division and Division of Engineering in Medicine, Brigham and Women’s Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts

ABSTRACT Senescent cells have undergone permanent growth arrest, adopt an altered secre- to induce growth arrest and senescence in tory , and accumulate in the kidney and other organs with and vitro, the key importance of short- injury. Senescence has diverse physiologic roles and experimental studies support eninginhumanagingislesswellestab- its importance in nephrogenesis, successful tissue repair, and in opposing malignant lished, and is not the focus of this review.5 transformation. However, recent murine studies have shown that depletion of Although the association between aging chronically senescent cells extends healthy lifespan and delays age-associated and senescence is well recognized,6,7 only disease—implicating senescence and the senescence-associated secretory phenotype recently have experiments depleting senes- as drivers of organ dysfunction. Great interest is therefore focused on the manipu- cent cells in murine models been shown to lation of senescence as a novel therapeutic target in kidney disease. In this review, postpone the onset of age-related diseases we examine current knowledge and areas of ongoing uncertainty regarding senes- and extend healthy lifespan, reigniting clin- cence in the human kidney and experimental models. We summarize evidence sup- ical and research interest.8–10 porting the role of senescence in normal kidney development and homeostasis but also senescence-induced maladaptive repair, renal fibrosis, and transplant failure. Initiation of Senescence Recent studies using senescent manipulation and depletion as novel therapies to Today, the term “cellular senescence” is treat renal disease are discussed, and we explore unanswered questions for future used to describe irreversible proliferative ar- research. rest with associated changes in organization, gene transcription, and pro- J Am Soc Nephrol 30: 726–736, 2019. doi: https://doi.org/10.1681/ASN.2018121251 tein secretion either in vitro or in vivo.11,12 Senescence can occur in multiple contexts across tissue and organ lifespan including Global demographics are changing, with WHAT IS SENESCENCE? physiologically appropriate “acute senes- the proportion of individuals over the cence” responses during injury, normal age of 65 in the United States predicted “Senescence” derives from the Latin organogenesis, tissue homeostasis, and re- to double within the next 25 years.1 Older “senex” meaning “old” and was origi- pair.13 In these settings senescence appears kidneys exhibit decreased function, altered nally used by biologists interchangeably to limit fibrosis before senescent cells are homeostasis, and increased susceptibility with the term “aging” to describe the de- removed by or immune clear- to pathologies including AKI, maladaptive cline in organism function with time. A ance.14 This form of senescence seems a repair, and the subsequent development seminal description by Leonard Hayflick tightly controlled, scheduled process. of CKD.2,3 In recent years, cellular senes- in 1961 of what is now recognized as cence has emerged as an important driver replicative senescence due to progressive of aging and age-related disease in multiple telomere shortening with prolonged cell Published online ahead of print. Publication date organs including the kidney. This review culture in vitro helped shape our modern available at www.jasn.org. examines our current understanding of understanding of the term. Senescence is Correspondence: Dr. David A. Ferenbach, Centre renal senescence, along with examples now recognized as both a response to for Inflammation Research, Queen’sMedicalRe- in vitro search Institute, University of Edinburgh, 47 Little from other organ systems where this high- prolonged culture or cell stress France Crescent, Edinburgh, EH16 4TJ, UK. Email: lights areas of potential research and trans- and as an important cellular stress and [email protected] in vivo 4 lational relevance to kidney homeostasis aging response . Although telo- Copyright © 2019 by the American Society of and disease. mere shortening is accepted as sufficient Nephrology

726 ISSN : 1046-6673/3005-726 J Am Soc Nephrol 30: 726–736, 2019 www.jasn.org REVIEW

Figure 1. Pathways to cellular senescence in eukaryotic cells. Multiple discrete cellular insults act via distinct signaling mechanisms to induce cell-cycle arrest in the kidney at either the G1/S (via inhibition of cdk2 and/or cdk4/6) or G2/M checkpoints (via Chk1/2 activation or cdc2/25c inhibition). Inactivation of and spindle/epigenetic/nucleolar stress trigger activation of the cyclin-dependent kinase inhibitor p16ink4a. Telomere shortening, DNA damage, mitogen or activation, and hypoxia/reoxygenation also result in G1/S cell-cycle arrest, via a pathway dependent on and p21cip1 activation. In contrast to this, developmental senescence appears to induce p21cip1 by pathways mediated by TGFb/PI3K and independent of p53. ATM/ATR/ARF, Ataxia–Telangiectasia Mutated/Ataxia Telangiectasia and Rad3-related protein/p14 Alternate Reading Frame (human).

In contrast, chronically senescent cells p21cip1 show increased cellular prolifer- Defining and Detecting accumulate in organs in response to a va- ation but also increased mortality after Senescent Cells riety of stressors including DNA damage ischemia/reperfusion injury, suggesting Whereas the physical and functional al- or critical telomere shortening leading to that close regulation of the is terations seen with senescence in vitro exposure of DNA ends,15 oncogenic mu- necessary for postinjury repair.18,19 are well characterized, identifying and tations, metabolic stress, mitochondrial Work with several experimental models characterizing senescent cells in vivo re- dysfunction, and inflammation.16,17 of fibrotic kidney disease identified mains challenging.23 This is due in part These insults trigger cell-cycle inhibition that accumulation of cells arrested at to the lack of any single feature uniquely and permanent cell-cycle arrest via path- the later G2/M checkpoint (via inhibi- identifying senescence, and difficulties in ways either dependent on or independent tion of cdc25c/cdc2 by p53 or activation assaying multiple attributes in individual of the DNA damage response (summa- of the Chk1 and Chk2 proteins) not only cells within a tissue.11,12,14 Important rizedinFigure1).Thesecellspersist results in senescence but triggers a pro- markers include senescence-associated within affected organs and are increas- fibrotic secretory phenotype.20–22 Inhib- b-galactosidase (SA-b-gal), p16ink4a,and ingly recognized as drivers of disease pro- iting this checkpoint using JNK, histone p21cip1; however, the presence/absence of gression rather than bystanders. deacetylase, or p53 inhibitors results in one or more of these factors is insufficient Most senescence-inducing stimuli reduced numbers of G2/M arrested cells to confirm senescence. The Van Deursen result in the induction of the cyclin- and reduced fibrosis in a model of uni- group has proposed that quantifying tis- dependent kinase inhibitors p16ink4a lateral renal ischemia.20 Delineating the sue senescence should comprise three ap- and/or p21cip1 which enhance check- differences and commonality between proaches: staining of cell-cycle arrest point activity, inducing cell-cycle arrest G1/S and G2/M arrested cells will be of markers, quantifying the effects of senes- at the G1/S cell-cycle checkpoint with interest in the future. Inhibition of cence-associated protein release, and ex- induction of the senescent pheno- movement of the cell through G1/S may, cluding coexisting cellular proliferation.12 type.11,12 Studies of experimental renal for example, reduce the number of cells that The generation of transgenic mice ex- disease in mice lacking p16ink4a and/or are arrested at G2/M. pressing genes driven by promoters

J Am Soc Nephrol 30: 726–736, 2019 Senescence and the Kidney 727 REVIEW www.jasn.org cloned from the Cdkn2a locus which en- preadipocytes intraperitoneally into that senescent cells are required at specific codes p16ink4a (INK-ATTAC, [LUC], young mice results in a 50-fold increase timepoints and in specific populations in and p16–3MR mice) represents a major in total body senescent cell number response to different injuries for optimal contribution to this field.24–26 Recent ad- compared with controls.8 Furthermore, repair.19,41 vances in single-cell sequencing should administration of serum from patients facilitate the identification and character- with chronic obstructive pulmonary dis- Defense ization of senescence on an individual cell ease (COPD) induces senescence in Senescence induction by oncogene acti- basis for the first time. bronchial epithelial cells in vitro,sug- vation is recognized as an important gesting that circulating factors may pro- physiologic mechanism preventing neo- The Senescence-Associated mote systemic senescence.37 plastic transformation. Although data in Secretory Phenotype human renal cancer are limited, one Despite exiting the cell cycle, senescent study showed that lowered expression cells remain metabolically active, PHYSIOLOGIC FUNCTIONS OF of senescence markers in human renal releasing a modified secretome into local SENESCENT CELLS cell carcinoma associates with worsening tissues which can significantly affect prognosis, whereas the antiproliferative neighboring cells and ultimately tissue Embryogenesis/Development effects of calcitriol on human renal can- function. This secretion of , Developmental senescence is a highly cer cells in vitro have been attributed to chemokines, growth factors, and prote- ordered and conserved cellular process induction of senescence.42,43 Oncogene ases is termed the senescence-associated observed in multiple species including tet- activation in murine hepatocytes drives secretory phenotype (SASP).27–29 SASP rapods and fish.38 The mesonephros and secretion of chemokines and cytokines components include IL-1b, IL-6, IL-8, endolymphatic sac of the human embryo resulting in immune-mediated clear- TGFb1, and WNT16B and these can contain senescent cells which affect tissue ance of these premalignant cells. Im- act in a paracrine and autocrine fashion remodeling and cell balance.13 Murine paired removal of senescent hepatocytes leading to persistence and propagation studies provide evidence that TGFb and results in subsequent development of of senescence within tissues.14,30–33 PI3K signaling activate p21cip1-induced se- hepatocellular carcinoma35 introducing Proinflammatory cytokines and che- nescence in vitro.13,39 Studies in mice show the concept of “senescent surveillance” mokines are relatively conserved SASP induction of SA-b-gal in the mesonephros as a defensive process, protecting against components across multiple organ in- simultaneous with falling proliferation malignant transformation by inducing juries.27 However, SASP composition markers immediately before involution cell-cycle arrest. The components of the may differ depending on the mode of se- of mesonephric tubules.13 Further studies SASP act to reinforce the growth arrest nescence induction and biologic context. show that growth arrest requires p21cip1 in neighboring cells, protecting an area Although direct evidence in the kidney is induction independent of p53, with mice of tissue from cancerous change.13,39 An- lacking, studies in other organ systems lacking p21cip1 exhibiting delayed meso- imal models where senescence induction provide a framework for future studies nephric regression but developing func- is blocked demonstrate increased suscep- in experimental renal disease. In repair tional kidneys—with increased apoptosis tibility to several .44,45 of cutaneous wounds in vivo, the secretion restoring appropriate cell number.13 of CYR61 (CCN1) is reported to be a key stimulus of fibroblast senescence, whereas Postinjury Repair SENESCENCE IN RENAL DISEASE SASP-derived PDGF-AA is required for Acute senescence is known to be impor- successful wound healing.24,34 In contrast, tant in successful scar formation, with Tubular epithelial cells are frequently im- immune clearance of oncogene-induced murine studies showing that senescent plicated in renal senescence, although senescent cells in the liver is promoted cell depletion after wounding in other cell types can exhibit positive strain- by CCL2 and chronically senescent cells vivo results in delayed wound closure— ing for markers of cell-cycle arrest. In a are reported to induce IL-6, plasminogen although eventual healing still occurs.24 series of human renal transplant biopsies, activator inhibitor 1 (PAI-1), and other Induction of fibroblast senescence con- all biopsy specimens showed p16ink4a diverse factors.10,35 The composition of trols the fibrotic pathways required for staining in the nuclei of distal tubules the SASP reflects both the origin of the normal healing,34 whereas activation of and collecting duct but staining was cell and the initiating stimuli, often initi- hepatic stellate cell senescence limits liver also present in podocytes, parietal epi- ated by NF-kB and p38 mitogen-activated fibrosis in a murine injury model.40 Inter- thelium of glomeruli, vascular smooth protein kinase signaling.36 estingly, p16ink4a knockout mice exposed muscle cells, and interstitial cells.46 Fur- The SASP provides an important to experimental renal injury show in- thermore, a series of patients with mechanism through which senescent creased epithelial proliferation and func- glomerular disease showed p16ink4a cells affect organ function via induction tional recovery after ischemia/reperfusion staining in a subset of glomerular, tubu- of paracrine senescence. Recent research injury but worsened fibrosis in unilateral lar, and interstitial cell nuclei; however, shows that transplanting senescent ureteric obstruction models, suggesting senescent tubular epithelial cells were

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AB C

DE F

Figure 2. Renal disease increases renal expression of the cyclin-dependent kinase inhibitor p16ink4a. (A) Normal kidney biopsy specimen from a 42-year-old living related donor with p16ink4a staining in some tubular epithelial cell nuclei.47 (B) Membranous nephropathy from a 64-year-old man demonstrating cytoplasmic and nuclear p16ink4a in tubular epithelium and interstitial nuclei (arrows).47 (C) FSGS biopsy specimen from a 48-year-old man, showing increased p16ink4a staining in some tubules and interstitial cell nuclei (arrows).47 (D) Grade 5 IgA nephropathy, with p16ink4a staining demonstrating increased cytoplasmic and nuclear staining.48 (E) Implantation biopsy specimen from a 45-year-old donor with expected low levels of p16ink4a-positive staining.46 (F) Repeat biopsy specimen of the same kidney 7 years post-transplantation demonstrating intense nuclear and cytoplasmic staining46 (note that the anti-p16ink4a [F12] antibody from Santa Cruz Biotechnology used in all of these images is no longer available). the key difference between disease and CKD shows reduced proteinuria, glomerular hy- control kidneys, present in 80% of cases Senescent cells are known to be present at pertrophy, and tubule-interstitial damage, compared with 21% in normal kid- increased levels in many renal diseases implicating senescence induction as contrib- neys.47 Additionally, a biopsy series of (Figure 2), and it appears likely that senes- uting to disease progression.53,54 patients with IgA nephropathy demon- cenceplaysaroleinthemaladaptiverepair strated increased p21cip1 and p16ink4a which contributes to many cases of progres- Autosomal Dominant Polycystic protein expression which was confined sive renal fibrosis after injury (Figure 3). Se- Kidney Disease to tubules.48 nescence, as indicated by p16ink4a-expressing In contrast to the above, autosomal Experimental data support the impor- cells, increases in patients with glomerular dominant polycystic kidney disease tance of the tubular epithelium. p16ink4a disease as compared with age-matched con- (ADPKD) is an example of senescence knockout mice have increased senescence trols.46 In IgA nephropathy, shorter telomere attenuating disease progression. Levels and subsequent fibrosis in tubular and in- lengths are found in mononuclear cells of p21cip1 are reduced in kidneys from terstitial cells after unilateral ureteric ob- extracted from urinary sediment collections human patients with ADPKD and in an- struction, most prominently in the collecting versus healthy controls, and a significant in- imal models of PKD.55 Treatment with duct.41 After ischemia reperfusion injury, verse correlation is shown between telo- the cyclin-dependent kinase (CDK) in- nuclear p21cip1 was localized to both distal mere length and serum creatinine.50 Recent hibitor roscovitine restores p21cip1 levels, and proximal nephron, including collect- bioinformatic analysis of both murine ex- increases SA-b-gal staining, decreases tu- ing ducts, with no glomerular staining perimental renal injury and human renal bular , and attenuates found.18 In Baker et al.’s26 seminal 2016 transplantation identifies significant upregu- disease progression in a murine model studyofablationofsenescentcellsin lation of Cdkn1a (p21cip1) in the aftermath of ADPKD.55,56 This suggests that a fail- INK-ATTACtransgenicmicedemon- of injury and in human transplanted kid- ure to induce senescence in ADPKD may strated senescence occurring in proximal neys undergoing AKI-to-CKD transition.51 contribute to cystic progression. tubules with increasing age. The impor- Finally, senescence may be a factor in pro- tance of the epithelial cell is supported by gression of diabetic nephropathy (DN). Kidney Transplantation in vitro data which demonstrate that a va- Levels of p16ink4a and SA-b-gal increase in Human studies have correlated levels of riety of injuries can induce renal proximal renal epithelial cells in biopsy specimens pre-transplant senescent cells in the kid- tubular epithelial cells into a senescent from patients with DN.52 Ap21cip1/p27kip1 ney with the subsequent development of phenotype.49 bigenic knockout mouse model of DN interstitial fibrosis, tubular atrophy, and

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Figure 3. Thepotential tissue effects of pathologic growth arrest in maladaptiverepair. This flow chart illustrates the putative steps required for repair after AKI. In the case of adaptive repair, acute senescence is induced but then cleared in a tightly controlled manner, with normal tissue structure being restored by proliferation of resident cells. In maladaptive repair, it is recognized that there is an accumulation of both tissue fibrosis and secondary senescent cells. Recent studies indicate that pharmacologic/genetic targeting of senescent cells may protect against progressive fibrosis in murine models of aging.26,71 chronic allograft nephropathy.46,47,57 KO mice developing less fibrosis when work has shown that, after irradiation, the Supporting this, elevated mRNA ex- receiving kidneys transplanted from generation of senescent hematopoietic pression of p21cip1 and p16ink4a in pre- p16ink4a KO mice and having signifi- stem cells results in immune dysfunc- transplant biopsy specimens correlates cantly higher graft survival, less tubu- tion, with selective depletion of these with poor outcomes in both experimen- lointerstitial damage, and higher tubular cells improving both stem cell and overall tal animals and man.58,59 In the setting epithelial cell proliferation scores.66 immune function in mice.70 Whether se- of renal transplantation, immune activa- nescence within the kidney or the im- tion and reduced immune clearance due and Senescent mune system interrupts the physiologic to immunosuppression may contribute Cell Clearance clearance of senescent cells and promotes to senescent cell accumulation.60,61 The Although accumulation of senescent renal disease remains an important un- interaction between senescent cells and cells with advancing age and with renal answered question. the post-transplant immune milieu is disease is well documented, whether this likely complex, because allograft rejection reflects (1) increased senescence induc- is associated with increased senescence tion, (2) altered SASP release preventing THERAPEUTIC TARGETING OF and glomerular, tubular, and interstitial immune clearance, or (3) a primary fail- SENESCENCE IN THE KIDNEY cells from rejected grafts express elevated ure within the itself re- levels of p16ink4a and p27kip1, correlat- mains largely unexplored. Although no Animal Models of Senescent Cell ing with the grade of chronic allograft direct studies exist on the role of the im- Deficiency or Depletion in the nephropathy.62 mune system in senescent cell clearance Kidney Senescence may explain some of the from the adult kidney, SASP release is Our understanding of senescent cell accu- effects of age on graft outcome. In hu- immunostumulatory35 and studies mulation and clearance in renal disease re- mans, increased numbers of senescent within the murine liver, uterus, and en- mains incomplete. Important lessons cells in an older donor kidney reduce dometrium have reported key roles for have been learned using wild-type and 1 the regenerative capacity of that trans- monocytes, , CD4 Tlym- transgenic mice to test the effect of de- planted kidney in response to injury.63 phocytes, and natural killer cells in the fective senescent cell induction on renal Grafts from older human donors are physiologic clearance of senescent development and disease.13,18,19,41,53,54 more vulnerable to ischemic injury and cells.35,67,68 Studies using Recent seminal studies demonstrate the more likely to develop delayed graft agents show that senescent cell ablation safety and efficacy of senescent cell de- function post-transplant.64,65 Murine results in reduced levels of inflammation pletion in vivo, using transgenic mice work supports a role for senescence in- in mice26 and in cells removed from in- with selective sensitivity to pharmacologic duction in graft outcome, with p16ink4a flamed human joints.69 Complementary agents,26,70 or wild-type mice treated with

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3,2019 736, Table 1. Experimental models of senescent cell de ciency/induction/depletion and their effects on renal outcomes Reference Renal Model Modulation of Senescence Renal Disease Outcome Effect of Intervention Baker et al.26 Natural aging in INK- INK-ATTAC 1AP20187 or vehicle ↑Glomerulosclerosis ↓Glomerulosclerosis ATTAC mice administration to deplete p16ink4a 1 ↑ß-gal positivity ↓ß-gal positivity cells Baar et al.71 Natural-aging p16-3MR FOXO4-DRI agent causes p53 nuclear ↑Serum urea FOXO4-DRI or GCV to p16-3MR admin mice and fast-aging Xpd exclusion. Ganciclovir treatment to ↓Serum urea TTD/TTD mice p163MR mice causes p16ink4a -positive ↓Lmnb1 expression ↑Lmnb1 expression restricted cell death ↑SASP expression (both XpdTTD/TTD and ↓SASP expression (both XpdTTD/TTD and aged aged p16-3MR) p16-3MR) Muñoz-Espín et al.13 Nephrogenesis WT versus P21cip1 KO mice with deficient ↓ß-gal positivity in P21cip1 KO mice in Use of PI3K inhibitor augments developmental growth arrest in nephrogenesis utero senescence in WT mice ↑Ki67 expression but apoptosis maintains development Wolstein et al.41 UUO WT versus p16ink4a KO mice with UUO induces ß-gal positivity, apoptosis, ↓ß-gal positivity impaired cell-cycle arrest and collagen deposition in WT mice ↓Apoptosis ↑Collagen ↑Proliferation after UUO in p16ink4a KO Megyesi et al.18 Renal IRI WT versus P21cip1 KO mice with impaired WT mice show tubular injury and raised ↑Proliferation cell-cycle arrest blood urea levels after IRI ↓Renal function ↑Mortality in P21cip1 KO Lee et al.19 Renal IRI WT versus p16ink4a /p19ARF double KO WT mice show marked p16ink4a and p16ink4a - and p19ARF -deficient mice show mice with impaired cell-cycle arrest p19ARF induction 28 d after IRI, with improved epithelial and microvascular repair, apoptosis and reduced tubular density with increased myeloid cell recruitment Al-Douahji et al.53 Diabetic WT versus p21cip1 KO WT mice develop albuminuria and Both p27kip1 KO and p21Cip1 KO mice were Wolf et al.54 Nephropathy WT versus p27kip1 KO glomerular hypertrophy protected from proteinuria and glomerular

eecneadteKidney the and Senescence expansion Braun et al.66 Renal transplant p16ink4a KO mice with impaired cell-cycle WT mice develop interstitial fibrosis and p16ink4a KO mice develop less atrophy and arrest tubular atrophy fibrosis after Tx fi Transgenic and genetic knockout mice have been used to study the effect of (1)de ciencies in the induction of senescence or (2) depletion of established senescent cells. Several of these models are summarized in www.jasn.org this table, with description of the experimental model of renal disease used, the alteration in senescence induction used, and any alterations in renal disease outcomes. TTD/TTD, /trichothiodystrophy; GCV, ganciclovir; WT, wild-type; KO, knock-out; UUO, unilateral ureteric obstruction; IRI, ischemia-reperfusion injury; Tx, Transplant. REVIEW 731 REVIEW www.jasn.org

Aging DNA damage Healthy Cell stress Kidney Oncogenes Senescent cells SASP release Oncosuppressors

JNK/p53 inhibitors cell cycle arrest Collagen

Myofibroblasts

Leukocytes Weight loss Sirolimus Metformin Exercise Secondary senescence PTBP1 shRNA Senolytic treatment SASP blockade Key to cell types FOXO4-DRI Tubular epithelia (TEC) ABT-263 Apoptotic/Necrotic TEC /Quercetin 1° Senescent TEC 17-DMAG 2° Senescent TEC Blocked Senescent TEC

Figure 4. Current and potential future interventions to target growth-arrested cells in the kidney in vivo. Multiple points in the generation of senescent cells are under investigation as potential windows to alter their accumulation and/or pathogenic effects. Interventions are being trialed to prevent senescent cell formation (e.g., weight loss, exercise), promote senescent cell apoptosis (e.g., FOXO4-DRI, ABT- 263), inhibit SASP release (e.g., sirolimus, metformin), or utilize the metabolic activity of senescent cells to activate compounds (e.g., galacto-oligosaccharide–conjugated drugs). shRNA, short hairpin RNA. drugs targeting antiapoptotic pathways mice and track and field athletes show that senescent cell apoptosis. Recent studies upregulated in senescent cells8,70,71 exercise associates with reduced markers have used this drug successfully to target (summarized in Table 1). These studies of senescence.73 In mice undergoing con- irradiation-induced senescence via the provide evidence that senescent cell trolled weight gain, animals switching to c-Jun/Bcl-xL/p21Cip1 pathway.70,75,76 depletion increases healthy lifespan reduced calorie intake reduce senescent With ABT-263 currently in phase 1 and by postponing the onset of several age- cell number in white .74 2 clinical trials for both hematologic and associated pathologies. Importantly, such solid organ malignancies there is poten- benefits have not come at the expense of tial for early clinical translation in non increased cancer incidence, indicating The INK-ATTAC and p16–3MR trans- malignant conditions.77,78 that depletion of chronically senescent genic mice use components of the Another combination includes dasa- cells differs from models lacking the Cdkn2a promoter (responsible for p16ink4a tinib (a tyrosine kinase inhibitor) and ability to induce senescence in response expression) to confer selective sensitiv- quercetin (a flavonoid) discovered using to DNA damage.26 Accordingly, there is ity of p16ink4a-expressing cells to phar- a bioinformatics-driven approach by major interest in the potential to manip- macologic depletion.24,26,70 Depletion Zhu et al.79 in 2015. Cultured senescent ulate senescent cell number or behavior to of chronically senescent cells from and nonsenescent cells were exposed to delay the onset and progression of numer- bothstrainsinagedorirradiatedani- compounds known to target senescent ous age-related diseases, including those mals benefits multiple organs including cell apoptotic pathways. Dasatinib and of the kidney (Figure 4). the kidney.26,70 Although informative, quercetin eliminate senescent but not the results of studies based exclusively nonsenescent cells in rodent and human Nonpharmacologic Approaches to upon such transgenes may not fully re- cell cultures, freshly isolated human tis- Reduce Senescent Cell capitulate the spectra of senescent cells sue explants, old mice, and mice with Accumulation seen in vivo, unless all such cells express accelerated ageing.8,79,80 Treating with Increased levels of physical activity and p16ink4a, which seems unlikely. dasatinib and quercetin alleviated the weight loss represent two nonpharmaco- Although the field of senolysis is in its physical dysfunction and frailty associ- logic approaches to reduce senescent cell infancy, several agents have generated ated with increased senescence. Mice generation. In mice exposed to a high-fat encouraging results in rodent models. lived longer and performed better in all diet, regimes of increased exercise reduce One such agent is ABT-263 (Navitoclax), measures of frailty tested.8 expression of senescence markers and a Bcl-2/w/xL inhibitor which targets a Other groups used an assay of SA- SASP release in adipose tissue.72 Studies dependence on the Bcl-2 pathway in se- b-gal expression to guide identification examining circulating leukocytes in both nescent cells, resulting in widespread of drugs capable of selectively targeting

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Figure 5. Unanswered questions about the roles and significance of senescent cells in the kidney. Several key questions regarding the role of the senescent cells in the kidney require ongoing research, with the goal of (1) quantifying senescent cell load noninvasively, (2)un- derstanding the need for senescent cells in renal repair after injury, (3) determining the factors preventing senescent cell clearance from the kidney, and (4) testing the efficacy of senescence-modifying interventions in man. senescence—leading to the identifica- removing the cells. Drugs familiar to ne- cells. Using this approach, PTRP1 was tion of the inhibitor 17DMAG as a phrologistsarebeing re-examined for this identified as a factor mediating the in- compound capable of increasing healthy purpose, including metformin and siro- flammatory effects of the SASP with lifespan and reducing p16ink4a-positive limus(rapamycin),bothofwhichactivate blockade inhibiting the proinflammatory cell number in a progeroid mouse and improve mitochondrial and tumorigenic properties of senescent model.81 Similarly, the increased ex- complex I function and can extend life- cells in the liver—a good example of ba- pression of SA-b-gal present within span.83–85 mTOR signaling is known to sic science identifying novel and drugga- diverse senescent cells has recently been be an important pathway in some forms ble targets for translation.81 harnessed to allow selective activation of of senescence86 and furthermore there cytotoxic compounds in vivo.Thisap- is some experimental evidence that siro- proach was used to target senescent cells limus itself inhibits the senescent cell THE FUTURE effectively in two models of cancer and phenotype.87–89 Given the previously fibrosis and represents an exciting avenue mentioned role of senescent cells in Fundamental questions remainsurround- for further clinical translation.82 Impor- wound healing, it is interesting to speculate ing the pathways regulating senescence, tant work by Baar et al.71 identified the whether a hitherto-unrecognized effect on their roles in tissue aging and dysfunc- FOXO4 protein as an important regula- senescence inhibition may contribute to tion, and their potential therapeutic use tor of p53 activity in senescent cells, and, the impaired wound healing associated (Figure 5). by designing an interfering peptide with clinical sirolimus use, although this “FOXO4-DRI,” demonstrated that selec- link remains unproven. How Can Senescent Cell Load Be tive apoptosis could be achieved in both Other candidate drugs include inhib- Determined In Vivo? aging- and chemotherapy-induced se- itors of IkB kinase, NF-kB, and the Janus Senescent cell burden in the kidney may nescent cells, with resultant improve- kinase pathway.90,91 Such approaches be a useful predictor of renal outcomes. ment in organismal fitness, including inhibit proinflammatory signaling Current means of detection are not suffi- protection of baseline renal structure pathways, such as NFkB or p38 mitogen- ciently specific and are often impractical and function. activated protein kinase pathways, becausestainedtissueisrequired.Whether disrupting SASP production.92,93 Are- increased senescent cell number on renal Senostatics cent study undertook an RNAi-based ap- biopsy predicts progression and func- Senostatics are drugs that inhibit elements proach to identify transcriptional factors tional decline better than existing markers of the senescent phenotype without mediating the SASP profile of senescent remains untested. Prospective studies

J Am Soc Nephrol 30: 726–736, 2019 Senescence and the Kidney 733 REVIEW www.jasn.org following patients with differing numbers damaged kidney. With better under- improve physical function and increase life- – ofsenescentcellswillberequiredtoanswer standing of their role in damage and span in old age. Nat Med 24: 1246 1256, 2018 9. Baker DJ, Perez-Terzic C, Jin F, Pitel KS, this question. Noninvasive serum or urine repair it may become possible to target Niederländer NJ, Jeganathan K, et al.: Op- of senescence would help re- them therapeutically, without compro- posing roles for p16Ink4a and p19Arf in se- searchers associate senescent burden with mising their essential regulatory func- nescence and ageing caused by BubR1 clinical , inform basic science tions in healing and cancer suppression. insufficiency. Nat Cell Biol 10: 825–836, 2008 research, and build a more complete un- 10. Baker DJ, Wijshake T, Tchkonia T, LeBrasseur NK, Childs BG, van de Sluis B, et al.: Clear- derstanding of their role in disease. ance of p16Ink4a-positive senescent cells ACKNOWLEDGMENTS delays ageing-associated disorders. Are Senescence Cells Important in 479: 232–236, 2011 Recovery? 11. Campisi J: Aging, cellular senescence, and The authors wish to thank Professor Jeremy – It is likely that optimal recovery post cancer. Annu Rev Physiol 75: 685 705, 2013 Hughes for his generous assistance with proof- 12. Childs BG, Durik M, Baker DJ, van Deursen renal injury requires the induction of reading of this manuscript. JM: Cellular senescence in aging and age- senescent cells at key timepoints, and a D.A.F. is supported by Intermediate Clinical related disease: From mechanisms to ther- delicate balance is needed to promote apy. Nat Med 21: 1424–1435, 2015 Fellowship WT100171MA from the Wellcome adaptive rather than maladaptive repair. 13. Muñoz-Espín D, Cañamero M, Maraver A, Trust. E.D.O. is supported by a Clinical Training The specificidentityandrolesofthese Gómez-López G, Contreras J, Murillo-Cuesta Fellowship from Kidney Research UK. JVB was S, et al.: Programmed cell senescence during cells in renal repair versus development fi supported by NIH DK072381 and DK039773. mammalian embryonic development. Cell of brosis remain unknown. 155: 1104–1118, 2013 DISCLOSURES 14. van Deursen JM: The role of senescent cells in ageing. Nature 509: 439–446, 2014 Why Do Senescent Cells J.V.B. is coinventor on KIM-1 patents that are 15. d’Adda di Fagagna F, Reaper PM, Clay- Accumulate with Age and Disease? assigned to Partners HealthCare and licensed to Farrace L, Fiegler H, Carr P, Von Zglinicki T, Johnson & Johnson, Sekisui, Novartis, Biogen Whether the accumulation seen in mul- et al.: A DNA damage checkpoint response Idec, R&D, and Astute. He is a consultant for As- tiple organs, including the kidney, with in telomere-initiated senescence. Nature tellas, Novartis, Roche, and Sekisui regarding the age reflects increased production or re- 426: 194–198, 2003 safety and efficacy of therapeutics or diagnostics for 16. Tchkonia T, Zhu Y, van Deursen J, Campisi J, duced immune clearance remains poorly AKI. He holds equity in MediBeacon, Sentien, and Kirkland JL: Cellular senescence and the se- understood. Intriguing studies from the Thrasos, and has grant support from Boehringer nescent secretory phenotype: Therapeutic Ingelheim and Roche. All other authors report no Fogo laboratory show that murine renal opportunities. JClinInvest123: 966–972, fi conflicts of interest. brosis is reduced in the aftermath of a 2013 young bone marrow transplant when 17. Campisi J, d’Adda di Fagagna F: Cellular compared with an older donor.94 The po- senescence: When bad things happen to REFERENCES – tential for therapies targeting immune good cells. Nat Rev Mol Cell Biol 8: 729 740, 2007 system aging and function to affect renal 1. 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