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Single-cell analysis of senescent epithelia reveals targetable mechanisms promoting fibrosis
Eoin D. O’Sullivan, … , Hassan Dihazi, David A. Ferenbach
Eoin D. O’Sullivan, … , Hassan Dihazi, David A. Ferenbach
Published November 22, 2022
Citation Information: JCI Insight. 2022;7(22):e154124. https://doi.org/10.1172/jci.insight.154124.
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Research Article Cell biology Nephrology

Single-cell analysis of senescent epithelia reveals targetable mechanisms promoting fibrosis

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Abstract

Progressive fibrosis and maladaptive organ repair result in significant morbidity and millions of premature deaths annually. Senescent cells accumulate with aging and after injury and are implicated in organ fibrosis, but the mechanisms by which senescence influences repair are poorly understood. Using 2 murine models of injury and repair, we show that obstructive injury generated senescent epithelia, which persisted after resolution of the original injury, promoted ongoing fibrosis, and impeded adaptive repair. Depletion of senescent cells with ABT-263 reduced fibrosis in reversed ureteric obstruction and after renal ischemia/reperfusion injury. We validated these findings in humans, showing that senescence and fibrosis persisted after relieved renal obstruction. We next characterized senescent epithelia in murine renal injury using single-cell RNA-Seq. We extended our classification to human kidney and liver disease and identified conserved profibrotic proteins, which we validated in vitro and in human disease. We demonstrated that increased levels of protein disulfide isomerase family A member 3 (PDIA3) augmented TGF-β–mediated fibroblast activation. Inhibition of PDIA3 in vivo significantly reduced kidney fibrosis during ongoing renal injury and as such represented a new potential therapeutic pathway. Analysis of the signaling pathways of senescent epithelia connected senescence to organ fibrosis, permitting rational design of antifibrotic therapies.

Authors

Eoin D. O’Sullivan, Katie J. Mylonas, Rachel Bell, Cyril Carvalho, David P. Baird, Carolynn Cairns, Kevin M. Gallagher, Ross Campbell, Marie Docherty, Alexander Laird, Neil C. Henderson, Tamir Chandra, Kristina Kirschner, Bryan Conway, Gry H. Dihazi, Michael Zeisberg, Jeremy Hughes, Laura Denby, Hassan Dihazi, David A. Ferenbach

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Figure 2

Senescent cell depletion following IRI results in decreased renal fibrosis.

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Senescent cell depletion following IRI results in decreased renal fibros...
(A) Schematic of murine senescent cell depletion following IRI experiment, with samples taken at days 0 and 28. Note senolytic ABT-263 administration for 2 weeks following injury. n = 6–8 per group. (B) Cystatin C measurement (ng/mL) — baseline group, mean cystatin C 377 (s.d. 84), and postnephrectomy group, mean 1,244 (s.d. 395). IRI and vehicle treatment group, mean 2,136 (s.d. 491), versus IRI and ABT-263 treatment, mean 1,286 (s.d. 372), 2-sided t test, P = 0.0006, difference 850, CI: 315–1,384. (C) Representative images and quantification of immunofluorescence and of Picrosirius red staining of kidneys. Quantification of total staining per renal cortex. * denotes P < 0.05. PDGFRB: uninjured 5.69% (s.d. 2.23), uninjured and ABT-263–treated 7.53% (s.d. 2.48). IRI and vehicle-treated 12.8% (s.d. 4.66) versus IRI and ABT-263–treated 14.3% (s.d. 2.82). ANOVA, P = 0.921 95% CI: –4.24–6.7. α-SMA: uninjured 0.84% (s.d. 0.33). uninjured and ABT-263–treated 1.56% (s.d. 0.39). IRI and vehicle-treated 25.5% (s.d. 23.4) versus IRI and ABT-263–treated 10.1% (s.d. 3.56). ANOVA, P = 0.108 95% CI: –33.74–2.92. Collagen 1: uninjured and vehicle-treated 3.75% (s.d. 1.73), uninjured and ABT-263–treated 1.95% (s.d. 0.74). UUO and vehicle-treated 12% (s.d. 12) versus UUO and ABT-263–treated 16.9% (s.d. 6.39). ANOVA, P = 0.921 95% CI: –4.2–6.75. Picrosirius red: control and vehicle-treated 0.98% (s.d. 0.37), control and ABT-263–treated 1.35% (s.d. 0.51). IRI and vehicle-treated 10.6% (s.d. 2.2) versus IRI and ABT-263–treated 5.6% (s.d. 1.14), difference 5.06%, (CI: 3.1–7.01) P < 0.005. For box plots, the center line represents the mean, the box limits the first and third quartiles, the whiskers ± 1.5 × IQR, and the points all the data. Scale bar = 100 μm.

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