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. 2021 Nov 8;13(11):e14146.
doi: 10.15252/emmm.202114146. Epub 2021 Nov 2.

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

Camille Cohen  1 , Océane Le Goff  1 , Frédéric Soysouvanh  2 , Florence Vasseur  1 , Marine Tanou  1 , Clément Nguyen  1 , Lucile Amrouche  1   3 , Julien Le Guen  4 , Oriana Saltel-Fulero  4 , Tanguy Meunier  4 , Thao Nguyen-Khoa  1   5 , Marion Rabant  1   6 , Dominique Nochy  7 , Christophe Legendre  1   3 , Gérard Friedlander  1 , Bennett G Childs  8 , Daren J Baker  8   9 , Bertrand Knebelmann  1   3 , Dany Anglicheau  1   3 , Fabien Milliat  2 , Fabiola Terzi  1
Affiliations

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

Camille Cohen et al. EMBO Mol Med. .

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.

Keywords: PAI-1; aging nephropathy; endothelial-podocyte cross-talk; kidney transplantation; senescence.

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Conflict of interest statement

B.G.C. and D.J.B. are shareholders and coinventors on patent applications licensed to or filed by Unity Biotechnology, a company developing senolytic medicines, including small molecules that selectively eliminate senescent cells. Research in the Baker laboratory has been reviewed by the Mayo Clinic Conflict of Interest Review Board and is being conducted in compliance with Mayo Clinic conflict of interest policies. All other authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. Physiological renal aging is characterized by glomerulosclerosis, podocyte loss, and glomerular endothelial senescence

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. p16 mRNA expression in whole kidney from young and old mice. n = 6 for young and old mice.

  8. mRNA expression of SASP components in kidneys from young and aged mice. n = 6 for young and old mice.

  9. 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.
Figure EV1
Figure EV1. Senescence affects endothelial cells in glomeruli and increases with age
  1. 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.

Figure EV2
Figure EV2. PAI‐1 is expressed in close vicinity to endothelial cells
  1. 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.

  2. 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.
Figure 2
Figure 2. Total body irradiation leads to glomerulosclerosis and glomerular endothelial cell senescence

  1. Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from irradiated (RT+) and non‐irradiated (RT−) control mice 12 months after irradiation. Original magnification ×400. Scale bar = 20 μm. n = 4 and n = 5 for RT+ and RT−, respectively.

  2. WT1 immunohistochemistry (upper panels) and quantification of WT1‐positive glomerular cells (lower panel) in kidneys from irradiated (RT+) and non‐irradiated (RT−) control mice 12 months after irradiation. Original magnification ×400. Scale bar = 20 μm. n = 5 for RT+ and RT− mice.

  3. Senescence‐associated β‐galactosidase staining (upper panels) and quantification of β‐galactosidase‐positive glomeruli (lower panel) in kidneys from irradiated (RT+) and non‐irradiated (RT−) control mice 12 months after irradiation. Original magnification ×400. Scale bar = 20 μm. n = 5 for RT+ and RT− mice.

  4. p16 mRNA expression in whole kidney from irradiated (RT+) and non‐irradiated (RT−) control mice 12 months after irradiation. n = 5 for RT+ and RT− mice.

  5. p21/griffonia simplicifolia/nephrin coimmunostaining (left panels) and quantification of endothelial p21‐positive cells (right panel) in glomeruli of kidneys from irradiated (RT+) and non‐irradiated (RT−) control mice 12 months after irradiation. Original magnification ×630. Scale bar = 10 μm. n = 4 for RT+ and RT− mice.

  6. 53BP1/CD34 coimmunostaining (left panels) and quantification of glomerular endothelial cell 53BP1 foci (right panel) in kidneys from irradiated (RT+) and non‐irradiated (RT−) control mice 12 months after irradiation. Original magnification ×630. Scale bar = 10 μm. n = 4 for RT+ and RT− mice. Arrows show 53BP1 foci.

  7. Relative mRNA expression of PAI‐1 in whole kidneys from irradiated (RT+) and non‐irradiated (RT−) control mice 12 months after irradiation. n = 5 for RT+ and RT− mice.

  8. PAI‐1 immunohistochemistry (left panels) and quantification of PAI‐1‐positive glomeruli (right panel) in kidneys from irradiated (RT+) and non‐irradiated (RT−) control mice 12 months after irradiation. Original magnification ×400. n = 4 for RT+ and RT− mice.

Data information: Data are means ± SEM. Statistical analysis: Student's t‐test: irradiated vs non‐irradiated mice.
Figure EV3
Figure EV3. Senescence affects endothelial cells in glomeruli of irradiated mice
p21/CD34 coimmunostaining (left panels) and quantification (right panel) in 12‐month‐old irradiated or non‐irradiated mice. Quantification represents the number of p21‐positive glomerular endothelial cells. 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.
Figure 3
Figure 3. A time course experiment that reveals progressive appearance of senescence and loss of podocytes with age

  1. WT1 (left upper panels) and p21 (left middle and lower panels) immunostaining in 4‐, 12‐, 18‐, and 24‐month‐old mice. Quantification of WT1‐positive cells (right upper panels), and glomerular and tubular p21‐positive cells (right lower panels). Original magnification ×400. Scale bar = 20 μm. n = 4 for each group. Arrows show positive p21 nuclei, either in glomeruli or in tubules.

  2. WT1 (left upper panels) and p21 (left middle and lower panels) immunostaining in 4‐ and 12‐month‐old irradiated (RT+) and non‐irradiated (RT−) mice. Quantification of WT1‐positive cells (right upper panels), and glomerular and tubular p21‐positive cells (right lower panels). Original magnification ×400. Scale bar = 20 μm. n = 4 for each group. Arrows show positive p21 nuclei, either in glomeruli or in tubules.

  3. Bioluminescence (BLI, upper panel) and quantification (lower panel) kidney BLI area in young or old mice (upper panel). n = 3 mice.

  4. Bioluminescence (BLI, upper panel) and quantification (lower panel) kidney BLI area in irradiated (RT+) and non‐irradiated (RT−) mice over time. n = 3–6 mice according to the different time points.

Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey–Kramer test.
Figure 4
Figure 4. Clearance of senescent cells prevents glomerulosclerosis, podocyte loss, and PAI‐1 overexpression during aging

  1. Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from 28‐month‐old p16 INK‐ATTAC mice treated with either vehicle (AP−) or AP20187 (AP+). Original magnification ×400. Scale bar = 20 μm. n = 5.

  2. WT1 immunostaining (upper panels) and quantification of WT1‐positive glomerular cells (lower panel) in 28‐month‐old p16 INK‐ATTAC mice treated with either vehicle (AP−) or AP20187 (AP+). Original magnification ×400. Scale bar = 20 μm. n = 5.

  3. PAI‐1 immunostaining (upper panels) and quantification of PAI‐1‐positive glomeruli (lower panel) in 28‐month‐old p16 INK‐ATTAC mice treated with either vehicle (AP−) or AP20187 (AP+). Original magnification ×400. Scale bar = 20 μm. n = 5.

Data information: Data are means ± SEM. Statistical analysis: Student's t‐test: AP+ vs AP− mice.
Figure 5
Figure 5. Endothelial‐specific PAI‐1 inactivation prevents age‐associated glomerular lesions

  1. PAI‐1 immunohistochemistry (upper panels) and quantification of PAI‐1‐positive glomeruli (lower panel) in kidneys of PAI‐1flox and PAI‐1Δendo mice at 22 months of age. Original magnification ×400. Scale bar = 20 μm. n = 4 for PAI‐1flox and PAI‐1Δendo mice.

  2. Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from PAI‐1flox and PAI‐1Δendo mice at 22 months of age. Original magnification ×400. Scale bar = 20 μm. n = 12 for PAI‐1flox and PAI‐1Δendo mice.

  3. TUNEL assay (upper panels) and quantification of TUNEL‐positive glomeruli (lower panel) in kidneys from PAI‐1flox and PAI‐1Δendo mice at 22 months of age. Original magnification ×400. Scale bar = 20 μm. n = 6 for PAI‐1flox and PAI‐1Δendo mice.

  4. WT1 immunostaining (upper panels) and quantification of WT1‐positive glomerular cells (lower panel) in kidneys from PAI‐1flox and PAI‐1Δendo mice at 22 months of age. Original magnification ×400. Scale bar = 20 μm. n = 4 and n = 6 for PAI‐1flox and PAI‐1Δendo mice, respectively.

  5. Serum creatinine measurement in PAI‐1flox (n = 6) and PAI‐1Δendo (n = 7) mice at 22 months of age.

  6. Senescence‐associated β‐galactosidase staining (upper panels) and quantification of β‐galactosidase‐positive glomeruli (lower panel) in kidneys from PAI‐1flox and PAI‐1Δendo mice at 22 months of age. Original magnification ×400. Scale bar = 20 μm. n = 4 for PAI‐1flox and PAI‐1Δendo mice.

  7. p21/griffonia simplicifolia/nephrin coimmunostaining in kidneys (upper panels) from PAI‐1flox and PAI‐1Δendo mice at 22 months of age and quantification (lower panel) of p21‐positive endothelial cells per glomeruli. Original magnification ×630. Scale bar = 10 μm. n = 4 and n = 6 for PAI‐1flox and PAI‐1Δendo mice, respectively.

  8. 53BP1/CD34 coimmunostaining (upper panels) and quantification of glomerular 53BP1 foci in glomerular endothelial cells from PAI‐1flox and PAI‐1Δendo mice at 22 months of age. Original magnification ×630. Scale bar = 10 μm. n = 4 and n = 6 for PAI‐1flox and PAI‐1Δendo mice, respectively. Arrows show 53BP1‐positive foci.

Data information: Data are means ± SEM. Statistical analysis: Student's t‐test: PAI‐1Δendo vs PAI‐1flox mice.
Figure 6
Figure 6. PAI‐1 secretion drives the endothelial cell–podocyte cross‐talk during senescence
  1. A

    Time of cell population doubling of primary glomerular endothelial cells from passage 8 (p8) to passage 17 (p17).

  2. B

    Senescence‐associated β‐galactosidase staining of glomerular endothelial cells (GEnC) at early (p9) or late (p17) passage.

  3. C

    Relative mRNA expression of senescence markers (p16 and p21) and SASP molecules (PAI‐1, IL‐6, IL‐8) in GEnC at early (p9) or late (p17) passage. n = 5 independent experiments.

  4. D

    Scheme of the experimental protocol used for coculture experiments with glomerular endothelial cells (GEnC) and podocytes.

  5. E

    Immunofluorescence of paxillin (green) and phalloidin (red) in podocytes stimulated with control medium, supernatant (SN) from glomerular endothelial cells from early (p9) or late (p17) passage and treated either with vehicle or with tiplaxtinin, a PAI‐1 inhibitor (upper panel). Quantification of focal adhesions (lower panel) was performed by counting the number of paxillin‐positive spots in 30 cells from three independent experiments. Original magnification ×1,000. Scale bar = 20 μm.

  6. F

    Annexin‐V‐FITC staining (upper panel) and quantification by FACS (lower panel) of podocytes stimulated with supernatant (SN) from GEnC at early (p9) or late (p17) passage and treated either with vehicle or with tiplaxtinin. n = 4 independent experiments.

Data information: Data are means ± SEM. Statistical analysis: Student's t‐test for (C); ANOVA followed by the Tukey–Kramer test for (E) and (F).
Figure EV4
Figure EV4. Recombinant PAI‐1 induces podocyte cytoskeleton modifications and detachment

  1. Immunofluorescence of paxillin (green) and phalloidin (red) on podocytes stimulated for 30 min with recombinant PAI‐1 at 5 nM. Quantification of focal adhesions (right panel) was performed by counting the number of paxillin‐positive spots in 30 cells from three independent experiments. Original magnification ×1,000. Scale bar = 20 μm.

  2. Podocyte morphology (left panels) and quantification of podocyte detachment (right panel) 30 min after stimulation by recombinant PAI‐1 at 5 nM. Original magnification ×400, n = 4 independent experiments.

Data information: Data are means ± SEM. Statistical analysis: Student's t‐test.
Figure 7
Figure 7. Glomerular PAI‐1 expression correlates with glomerular senescence and predicts outcomes of kidney transplant from elderly donors

  1. PAI‐1 staining (left panel) and quantification (right panel) in glomeruli of kidney biopsies from young (< 40 years of age, n = 8) or old (> 80 years of age, n = 10) kidney donors at time of transplantation (M0). Original magnification ×400. Scale bar = 20 μm.

  2. p16 staining (left panel) and quantification (right panel) in glomeruli of kidney biopsies from young (< 40 years of age, n = 8) or old (> 80 years of age, n = 10) kidney donors at time of transplantation (M0). The arrows point to the staining. Original magnification ×400. Scale bar = 20 μm.

  3. PAI‐1/CD34 coimmunostaining in old‐positive or old‐negative glomerular PAI‐1 donors. Original magnification ×630. Scale bar = 10 μm.

  4. p16/CD34 coimmunostaining in old‐positive glomerular PAI‐1 donors. Original magnification ×630. Scale bar = 10 μm.

  5. Representation of PAI‐1 and p16 staining results at time of transplantation in young (n = 8) or old donors (n = 10). Each line represents one patient. Green color is used for negative staining, and red color, for positive staining.

  6. Quantification of the glomerulosclerosis score at time of (M0), or 12 months after (M12), kidney transplantation from elderly donors in patients displaying a negative (PAI‐1−, n = 17) or positive (PAI‐1+, n = 13) glomerular PAI‐1 staining at time of transplantation (M0).

  7. Estimated glomerular filtration rate (eGFR) at time of (M0), or 12 months after (M12), kidney transplantation in patients displaying a negative (PAI‐1−, n = 17) or positive (PAI‐1+, n = 18) glomerular PAI‐1 staining at time of transplantation (M0).

  8. Urinary PAI‐1 measured by ELISA in a cohort of elderly patients with (n = 48) or without (n = 48) chronic kidney disease.

Data information: Data are means ± SEM. Statistical analysis: Student's t‐test for (A), (B), and (H), ANOVA followed by the Tukey–Kramer test for (F) and (G).
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