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Glomerular endothelial cell senescence drives age‐related kidney disease through PAI‐1

2021; Springer Nature; Volume: 13; Issue: 11 Linguagem: Inglês

10.15252/emmm.202114146

1757-4684

Camille Cohen, O. Le Goff, Frédéric Soysouvanh, Florence Vasseur, Marine Tanou, Clément Nguyen, Lucile Amrouche, J. Le Guen, Oriana Saltel‐Fulero, Tanguy Meunier, Thao Nguyen‐Khoa, Marion Rabant, Dominique Nochy, Christophe Legendre, Gérard Friedlander, Bennett G. Childs, Darren J. Baker, Bertrand Knebelmann, Dany Anglicheau, Fabien Milliat, Fabiola Terzi,

Renal and related cancers

Article2 November 2021Open Access Transparent process Glomerular endothelial cell senescence drives age-related kidney disease through PAI-1 Camille Cohen Camille Cohen Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Search for more papers by this author Océane Le Goff Océane Le Goff Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Search for more papers by this author Frédéric Soysouvanh Frédéric Soysouvanh Institut de Radioprotection et de Sureté Nucléaire (IRSN), Laboratoire Radiobiologie des Expositions Médicale, Fontenay-aux-Roses, France Search for more papers by this author Florence Vasseur Florence Vasseur Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Search for more papers by this author Marine Tanou Marine Tanou Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Search for more papers by this author Clément Nguyen Clément Nguyen Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Search for more papers by this author Lucile Amrouche Lucile Amrouche Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Service de Néphrologie-Transplantation, Hôpital Necker Enfants Malades, AP-HP centre, Université de Paris, Paris, France Search for more papers by this author Julien Le Guen Julien Le Guen Service de Gériatrie, Hôpital Européen Georges Pompidou, AP-HP Centre, Université de Paris, Paris, France Search for more papers by this author Oriana Saltel-Fulero Oriana Saltel-Fulero Service de Gériatrie, Hôpital Européen Georges Pompidou, AP-HP Centre, Université de Paris, Paris, France Search for more papers by this author Tanguy Meunier Tanguy Meunier Service de Gériatrie, Hôpital Européen Georges Pompidou, AP-HP Centre, Université de Paris, Paris, France Search for more papers by this author Thao Nguyen-Khoa Thao Nguyen-Khoa orcid.org/0000-0003-3738-7299 Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Service de Biochimie, Hôpital Necker Enfants Malades, AP-HP Centre, Université de Paris, Paris, France Search for more papers by this author Marion Rabant Marion Rabant Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Service d'Anatomo-Pathologie, AP-HP, Hôpital Necker Enfants Malades, AP-HP Centre, Université de Paris, Paris, France Search for more papers by this author Dominique Nochy Dominique Nochy Service d'Anatomo-Pathologie, Hôpital Européen George Pompidou, AP-HP Centre, Université de Paris, Paris, France Search for more papers by this author Christophe Legendre Christophe Legendre Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Service de Néphrologie-Transplantation, Hôpital Necker Enfants Malades, AP-HP centre, Université de Paris, Paris, France Search for more papers by this author Gérard Friedlander Gérard Friedlander Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Search for more papers by this author Bennett G Childs Bennett G Childs Department of Pediatrics, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Daren J Baker Daren J Baker orcid.org/0000-0001-9006-1939 Department of Pediatrics, Mayo Clinic College of Medicine, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Bertrand Knebelmann Bertrand Knebelmann Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Service de Néphrologie-Transplantation, Hôpital Necker Enfants Malades, AP-HP centre, Université de Paris, Paris, France Search for more papers by this author Dany Anglicheau Dany Anglicheau Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Service de Néphrologie-Transplantation, Hôpital Necker Enfants Malades, AP-HP centre, Université de Paris, Paris, France Search for more papers by this author Fabien Milliat Fabien Milliat orcid.org/0000-0001-8249-9498 Institut de Radioprotection et de Sureté Nucléaire (IRSN), Laboratoire Radiobiologie des Expositions Médicale, Fontenay-aux-Roses, France Search for more papers by this author Fabiola Terzi Corresponding Author Fabiola Terzi [email protected] orcid.org/0000-0001-6107-8125 Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Search for more papers by this author Camille Cohen Camille Cohen Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Search for more papers by this author Océane Le Goff Océane Le Goff Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Search for more papers by this author Frédéric Soysouvanh Frédéric Soysouvanh Institut de Radioprotection et de Sureté Nucléaire (IRSN), Laboratoire Radiobiologie des Expositions Médicale, Fontenay-aux-Roses, France Search for more papers by this author Florence Vasseur Florence Vasseur Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Search for more papers by this author Marine Tanou Marine Tanou Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Search for more papers by this author Clément Nguyen Clément Nguyen Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Search for more papers by this author Lucile Amrouche Lucile Amrouche Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Service de Néphrologie-Transplantation, Hôpital Necker Enfants Malades, AP-HP centre, Université de Paris, Paris, France Search for more papers by this author Julien Le Guen Julien Le Guen Service de Gériatrie, Hôpital Européen Georges Pompidou, AP-HP Centre, Université de Paris, Paris, France Search for more papers by this author Oriana Saltel-Fulero Oriana Saltel-Fulero Service de Gériatrie, Hôpital Européen Georges Pompidou, AP-HP Centre, Université de Paris, Paris, France Search for more papers by this author Tanguy Meunier Tanguy Meunier Service de Gériatrie, Hôpital Européen Georges Pompidou, AP-HP Centre, Université de Paris, Paris, France Search for more papers by this author Thao Nguyen-Khoa Thao Nguyen-Khoa orcid.org/0000-0003-3738-7299 Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Service de Biochimie, Hôpital Necker Enfants Malades, AP-HP Centre, Université de Paris, Paris, France Search for more papers by this author Marion Rabant Marion Rabant Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Service d'Anatomo-Pathologie, AP-HP, Hôpital Necker Enfants Malades, AP-HP Centre, Université de Paris, Paris, France Search for more papers by this author Dominique Nochy Dominique Nochy Service d'Anatomo-Pathologie, Hôpital Européen George Pompidou, AP-HP Centre, Université de Paris, Paris, France Search for more papers by this author Christophe Legendre Christophe Legendre Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Service de Néphrologie-Transplantation, Hôpital Necker Enfants Malades, AP-HP centre, Université de Paris, Paris, France Search for more papers by this author Gérard Friedlander Gérard Friedlander Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Search for more papers by this author Bennett G Childs Bennett G Childs Department of Pediatrics, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Daren J Baker Daren J Baker orcid.org/0000-0001-9006-1939 Department of Pediatrics, Mayo Clinic College of Medicine, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Bertrand Knebelmann Bertrand Knebelmann Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Service de Néphrologie-Transplantation, Hôpital Necker Enfants Malades, AP-HP centre, Université de Paris, Paris, France Search for more papers by this author Dany Anglicheau Dany Anglicheau Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Service de Néphrologie-Transplantation, Hôpital Necker Enfants Malades, AP-HP centre, Université de Paris, Paris, France Search for more papers by this author Fabien Milliat Fabien Milliat orcid.org/0000-0001-8249-9498 Institut de Radioprotection et de Sureté Nucléaire (IRSN), Laboratoire Radiobiologie des Expositions Médicale, Fontenay-aux-Roses, France Search for more papers by this author Fabiola Terzi Corresponding Author Fabiola Terzi [email protected] orcid.org/0000-0001-6107-8125 Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France Search for more papers by this author Author Information Camille Cohen1, Océane Le Goff1, Frédéric Soysouvanh2, Florence Vasseur1, Marine Tanou1, Clément Nguyen1, Lucile Amrouche1,3, Julien Le Guen4, Oriana Saltel-Fulero4, Tanguy Meunier4, Thao Nguyen-Khoa1,5, Marion Rabant1,6, Dominique Nochy7, Christophe Legendre1,3, Gérard Friedlander1, Bennett G Childs8, Daren J Baker8,9, Bertrand Knebelmann1,3, Dany Anglicheau1,3, Fabien Milliat2 and Fabiola Terzi *,1 1Université de Paris, INSERM U1151, CNRS UMR 8253, Institut Necker Enfants Malades (INEM), Département “Croissance et Signalisation”, Paris, France 2Institut de Radioprotection et de Sureté Nucléaire (IRSN), Laboratoire Radiobiologie des Expositions Médicale, Fontenay-aux-Roses, France 3Service de Néphrologie-Transplantation, Hôpital Necker Enfants Malades, AP-HP centre, Université de Paris, Paris, France 4Service de Gériatrie, Hôpital Européen Georges Pompidou, AP-HP Centre, Université de Paris, Paris, France 5Service de Biochimie, Hôpital Necker Enfants Malades, AP-HP Centre, Université de Paris, Paris, France 6Service d'Anatomo-Pathologie, AP-HP, Hôpital Necker Enfants Malades, AP-HP Centre, Université de Paris, Paris, France 7Service d'Anatomo-Pathologie, Hôpital Européen George Pompidou, AP-HP Centre, Université de Paris, Paris, France 8Department of Pediatrics, Mayo Clinic College of Medicine, Rochester, MN, USA 9Robert and Arlene Kogod Center on Aging, Mayo Clinic College of Medicine, Rochester, MN, USA *Corresponding author. Tel: +33 1 40 61 53 64; E-mail: [email protected] EMBO Mol Med (2021)13:e14146https://doi.org/10.15252/emmm.202114146 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The mechanisms underlying the development of glomerular lesions during aging are largely unknown. It has been suggested that senescence might play a role, but the pathophysiological link between senescence and lesion development remains unexplained. Here, we uncovered an unexpected role for glomerular endothelial cells during aging. In fact, we discovered a detrimental cross-talk between senescent endothelial cells and podocytes, through PAI-1. In vivo, selective inactivation of PAI-1 in endothelial cells protected glomeruli from lesion development and podocyte loss in aged mice. In vitro, blocking PAI-1 in supernatants from senescent endothelial cells prevented podocyte apoptosis. Consistently, depletion of senescent cells prevented podocyte loss in old p16 INK-ATTAC transgenic mice. Importantly, these experimental findings are relevant to humans. We showed that glomerular PAI-1 expression was predictive of poor outcomes in transplanted kidneys from elderly donors. In addition, we observed that in elderly patients, urinary PAI-1 was associated with age-related chronic kidney disease. Altogether, these results uncover a novel mechanism of kidney disease and identify PAI-1 as a promising biomarker of kidney dysfunction in allografts from elderly donors. SYNOPSIS Kidneys develop lesions with age, and in particular glomerulosclerosis, but the molecular mechanisms involved in the deterioration process are unclear. Here, an unexpected role for glomerular endothelial cells during aging was uncovered. Senescent glomerular endothelial cells increased with age, whereas the number of podocytes decreased. The existence of a detrimental crosstalk between senescent glomerular endothelial cells and podocyte was demonstrated in vivo and in vitro. Depletion of senescent cells prevented podocyte loss with age. PAI-1 was a critical mediator of this cross-stalk, and its selective inactivation in endothelial cell preserved kidneys from glomerulosclerosis during aging. PAI-1 immunostaining predicted kidney allograft dysfunction after transplantation from elderly donors. PAI-1 excretion was increased in the urine of elderly patients with recognized aging nephropathy. The paper explained Problem As the worldwide population ages, age-related organ diseases become a real public health burden. Due to the physiological reduction in the number of functional nephrons with age, the prevalence of chronic kidney disease (CKD) dramatically increases in elderly people. CKD is characterized by the inexorable progression toward end-stage renal disease (ESRD). The survival and quality of life of ESRD patients are very poor. Therefore, there is an urgent need to understand the pathophysiological processes leading to CKD in elderly people in order to develop efficient therapeutic strategies. Results Here, we identified PAI-1 as a critical mediator of a detrimental cross-talk between senescent endothelial cells and podocytes that leads to the development of renal lesions. Deletion of PAI-1 in endothelial cells or clearance of senescent endothelial cells protected transgenic mice from podocyte loss and glomerulosclerosis with age. More importantly, we demonstrated that these data have a potential great medical impact. In fact, we identified PAI-1 immunostaining on preimplantation biopsies as an extremely early sensitive biomarker able to predict the suitability of kidneys from elderly donors for transplantation. Impact As organ shortage for kidney transplantation is a major public health problem, with thousands of people dying each year while waiting for a kidney transplant, the criteria for organ supply have been expanded leading to the consideration of organs from older donors. In this respect, our study provides an important contribution. In fact, we identified PAI-1 as a novel biomarker, ready to be used in clinical practice and able to predict the survival of kidney allografts from elderly donors. Introduction Chronic kidney disease (CKD) is one of the major public health challenges of the 21st century. Despite efforts from the healthcare community, the survival and quality of life of end-stage renal disease (ESRD) patients remain severely impaired. CKD is characterized by the progressive decline of renal function to ESRD that occurs once a critical number of nephrons have been lost, irrespective of the cause of renal damage (Hostetter et al, 1982). Due to the physiological reduction in functional nephrons with age, the prevalence of CKD dramatically increases in elderly people. Recent studies report that CKD affects 15–38% of people older than 65 years, and rises up to 50% in people older than 85 years (Coresh et al, 2007; Hill et al, 2016). Hence, there is an urgent need to elucidate the mechanisms underlying the progression of CKD in elderly people in order to develop efficient therapeutic strategies. Age-related kidney diseases are characterized by the development of glomerular lesions, such as glomerulosclerosis and podocyte loss (Steffes et al, 2001; Appel et al, 2009). In living kidney donors, selected because of normal renal function and the absence of any disease susceptible to alter kidney function such as diabetes or hypertension, the percentage of glomerulosclerosis increases with age. In particular, up to 82% of patients older than 70 years display glomerulosclerosis compared with 19% of donors between 18 and 29 years of age (Rule et al, 2010). Although this association seems to be robust, the cellular and molecular mechanisms leading to the development of glomerulosclerosis remain unclear. It has been proposed that oxidative stress and ischemia-induced activation of pro-inflammatory and pro-fibrotic pathways may play a role (Valentijn et al, 2018). Recently, it has been suggested that cellular senescence may also be detrimental during aging. With the use of a very elegant transgenic mouse model (p16 INK-ATTAC), where senescent cells die by apoptosis as soon as they express p16 upon AP20187 administration, Baker et al (2016) have shown that systemic depletion of senescent cells delayed age-related organ phenotypes, including glomerulosclerosis. It is worth noting that the number of senescent cells increases in kidneys of elderly people (Melk et al, 2004). Intriguingly, however, cellular senescence seems to occur mainly in the tubular compartment, while lesions affect almost exclusively the glomerular compartment during aging (Krishnamurthy et al, 2004; Melk et al, 2004; Baker et al, 2016). Senescence describes a cellular process in which a cell undergoes a permanent cell cycle arrest mediated through the cyclin-dependent kinase inhibitors, p16 and p21 (Salama et al, 2014). Despite the arrest of the cell cycle, senescent cells remain viable and metabolically active and secrete several molecules, known as the senescence-associated secretory phenotype (SASP). SASP includes pro-inflammatory cytokines, growth factors, proteases, and extracellular matrix components (Kuilman et al, 2008; Salama et al, 2014; Watanabe et al, 2017). These molecules play a critical role during development and wound healing, but they are also detrimental in pathological processes by favoring a local pro-fibrotic and pro-inflammatory environment (Kuilman et al, 2008; Childs et al, 2015; Watanabe et al, 2017). Interestingly, several of these molecules, such as PAI-1, IL-6, or TGF-β, have been shown to play a role in the development of glomerular lesions in several experimental models of CKD (Ding et al, 2001; Eddy & Fogo, 2006; Mei & Zheng, 2009). However, since none of the molecules composing the SASP is specific to SASP, it is difficult to determine whether their expression indicates that senescence is involved in this pathological setting or, on the contrary, testifies to the existence of a more general lesional process. Senescence can be triggered by different stimuli (Kuilman et al, 2010). The most classical is the shortening of telomeres observed during aging. Due to the absence of specific repair machinery, telomeres shorten at each cell division. When they reach a critical size, the DNA damage response (DDR) is activated leading to senescence (d'Adda di Fagagna et al, 2003; Kuilman et al, 2010). By favoring persistent DDR, chemotherapy or X-rays are also very well-known inducers of senescence (d'Adda di Fagagna, 2008). Accordingly, irradiation is a widely used technique to induce senescence in experimental in vivo and in vitro models (Nguyen et al, 2018). Taking all these data together, we hypothesize that senescence might drive age-related glomerular lesions in a non-cell-autonomous way by the secretion of detrimental components of SASP. To investigate this hypothesis, we combined experimental models of aging and genetically modified mice with in vitro experiments and human samples. We demonstrated the key role played by endothelial senescence in podocyte loss and subsequent lesion development. Mechanistically, we identified PAI-1 as a crucial mediator of the interaction between endothelial cells and podocytes. More importantly, we show that these data are relevant to humans and identified PAI-1 as a promising biomarker for predicting allograft function from elderly donors, as well as kidney function in elderly individuals. Results Aging nephropathy is associated with glomerular endothelial cell senescence In order to investigate the role of senescence in aging nephropathy, we first compared kidney morphology of 24-month-old mice with 4-month-old mice. We confirmed that 24-month-old mice developed glomerulosclerosis (Fig 1A) together with podocyte loss (Fig 1B). Podocyte loss was associated with an increase in glomerular apoptosis, as revealed by TUNEL assay (Fig 1C). Figure 1. Physiological renal aging is characterized by glomerulosclerosis, podocyte loss, and glomerular endothelial senescence Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from young and aged mice. Original magnification ×400. Scale bar = 20 μm. n = 6 for young and old mice. WT1 immunohistochemistry (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from young and aged mice. Original magnification ×400. Scale bar = 20 μm. n = 6 for young and old mice. TUNEL assay (upper panels) and quantification of TUNEL-positive tubular cells (upper panel) in glomeruli from young and aged mice. Panels are representative images of 6 young and old mice. Scale bar = 20 μm. Senescence-associated β-galactosidase staining (upper panels) and quantification of β-galactosidase-positive glomeruli (lower panel) in kidneys from young and aged mice. Original magnification ×400. Scale bar = 20 μm. Panels are representative images of four young and old mice. p21/griffonia simplicifolia/nephrin coimmunostaining in kidneys from young and aged mice (left panels) and quantification (right panel) of p21-positive endothelial cells per glomeruli. Original magnification ×630. Scale bar = 10 μm. Panels are representative images of 6 young and old mice. 53BP1/CD34 coimmunostaining in kidneys from young and aged mice (left panels) and quantification (right panels) of 53BP1 foci per glomerular endothelial cell. Original magnification ×630. Scale bar = 10 μm. Panels are representative images of 6 young and old mice. p16 mRNA expression in whole kidney from young and old mice. n = 6 for young and old mice. mRNA expression of SASP components in kidneys from young and aged mice. n = 6 for young and old mice. PAI-1 immunohistochemistry (left panels) and quantification of PAI-1-positive glomeruli (right panel) in kidneys from young and aged mice. Original magnification ×400. Scale bar = 20 μm. n = 5 for young and old mice. Data information: Data are means ± SEM. Statistical analysis: Student's t-test: young vs old mice. Download figure Download PowerPoint Senescent cells display several characteristics that favor their identification, such as increased lysosomal biosynthesis, cell cycle arrest, or persistence of DNA damage (Kuilman et al, 2010). Specific markers can identify each of these events, such as senescence-associated β-galactosidase (SA-βGal), p16 and p21, or p53 binding protein 1 (53BP1). Interestingly, senescent cells were identified mainly in glomeruli of old mice. In fact, SA-βGal staining was found in 49% of glomeruli in old mice compared with 17% in young mice (Fig 1D). Similarly, immunofluorescence studies using anti-p21, anti-53BP1, and anti-pH2AX antibodies confirmed the presence of senescent cells in glomeruli from old mice (Figs 1E and F, and EV1A). The mRNA level of p16 was also significantly increased in old mice as compared to young mice (Fig 1G). Glomeruli are composed of different cell types, i.e., endothelial cells, mesangial cells, and podocytes. To characterize which cell type undergoes senescence during aging, we performed coimmunostaining with antibodies directed against p21, a senescence marker, nephrin, a podocyte marker, and griffonia simplicifolia, a lectin that recognizes endothelial cells. Results showed that p21 colocalizes with griffonia simplicifolia, but not with nephrin (Fig 1E). In addition, we observed that the number of p21-positive cells increased in aged mice as compared to young animals (Fig 1E). To confirm that senescence affects mainly endothelial cells in glomeruli, we performed additional coimmunostaining between the senescence markers, 53BP1 or pH2AX, and the endothelial markers, CD34 or griffonia simplicifolia. We confirmed an increase in endothelial senescence in glomeruli of old mice (Figs 1F and EV1A). Click here to expand this figure. Figure EV1. Senescence affects endothelial cells in glomeruli and increases with age A, B. pH2AX/griffonia simplicifolia coimmunostaining (left panels) and quantification (right panel) in kidneys from (A) young and aged mice and (B) 12-month-old irradiated and non-irradiated mice. Quantification represents the number of pH2AX foci per glomerular endothelial cell. Original magnification ×630. Scale bar = 10 μm. Panels are representative images of four mice in each group. Data are means ± SEM. Statistical analysis: Student's t-test. Download figure Download PowerPoint Glomerular endothelial cell senescence is associated with SASP The observation that senescence occurred mainly in endothelial cells and apoptosis occurred mainly in podocytes suggests a cross-talk between these cells. Senescent cells are characterized by the secretion of several molecules, grouped under the name of SASP (Salama et al, 2014), suggesting that one of these molecules might drive this cross-talk. We observed that several components of SASP known to play a potential role in glomerulosclerosis (Wang et al, 2017), such as PAI-1, IL-1β, IL-6, or MMP13, were upregulated in kidneys of old mice as compared to young mice (Fig 1H). In contrast, the expression of VEGF-A was decreased (Fig 1H). Since a number of previous studies showed that the increase in PAI-1 expression is associated with the development of glomerular lesions in several experimental models of glomerular diseases (Rondeau et al, 1990; Yamamoto et al, 1993; Grandaliano et al, 2000; Lee et al, 2001; Hamano et al, 2002; Paueksakon et al, 2002), we decided to first focus on this paracrine mediator. Immunohistochemical experiments confirmed the increased expression of PAI-1 protein in old mice, in which 42% of glomeruli were PAI-1-positive compared with 4% in young mice (Fig 1I). More importantly, by performing coimmunostaining experiments, we demonstrated that PAI-1 is expressed in close vicinity to endothelial cells (Fig EV2A). The fact that PAI-1 is a secreted protein may account for not perfectly overlapping with griffonia simplicifolia staining. Click here to expand this figure. Figure EV2. PAI-1 is expressed in close vicinity to endothelial cells A, B. PAI-1/griffonia simplicifolia coimmunostaining in kidneys from (A) young and aged mice and (B) 12-month-old irradiated and non-irradiated mice. C. PAI-1/griffonia simplicifolia coimmunostaining in kidneys from 22-month-old PAI-1flox and PAI-1Δendo mice. Original magnification ×630. Data information: Scale bar = 10 μm. Panels are representative images of four mice in each group. Download figure Download PowerPoint To confirm the possible relationship between senescence and glomerulosclerosis, we studied mice subjected to sublethal total body irradiation, since irradiation is known to induce both senescence and glomerular lesions (Dawson et al, 2010; Nguyen et al, 2018). As expected, irradiated mice developed glomerulosclerosis (Fig 2A), podocyte loss (Fig 2B), and increased senescence, 12 months after irradiation (Fig 2C and D). Interestingly, p21/CD34, 53BP1/CD34, and pH2AX/griffonia simplicifolia colocalization experiments revealed that senescence affected mainly endothelial cells in glomeruli (Figs 2F, EV1B, and EV3). Similarly, a triple staining with p21/nephrin/griffonia simplicifolia confirmed that p21 is expressed in endothelial cells, but not in podocytes (Fig 2E). PAI-1 mRNA and protein expressions were also significantly higher in kidneys of irradiated mice as compared to control non-irradiated ones (Fig 2G and H). Costaining experiments revealed that PAI-1 is expressed in close vicinity to endothelial cells (Fig EV2B). Toget