Polyploid superficial uroepithelial bladder barrier cells express features of cellular senescence across the lifespan and are insensitive to senolytics

Abstract

Lower urinary tract dysfunction (LUTD) increases with aging. Ensuing symptoms including incontinence greatly impact quality of life, isolation, depression, and nursing home admission. The aging bladder is hypothesized to be central to this decline, however, it remains difficult to pinpoint a singular strong driver of aging-related bladder dysfunction. Many molecular and cellular changes occur with aging, contributing to decreased resilience to internal and external stressors, affecting urinary control and exacerbating LUTD. In this study, we examined whether cellular senescence, a cell fate involved in the etiology of most aging diseases, contributes to LUTD. We found that umbrella cells (UCs), luminal barrier uroepithelial cells in the bladder, show senescence features over the mouse lifespan. These polyploid UCs exhibit high cyclin D1 staining, previously reported to mediate tetraploidy-induced senescence in vitro. These senescent UCs were not eliminated by the senolytic combination of Dasatinib and Quercetin. We also tested the effect of a high-fat diet (HFD) and senescent cell transplantation on bladder function and showed that both models induce cystometric changes similar to natural aging in mice, with no effect of senolytics on HFD-induced changes. These findings illustrate the heterogeneity of cellular senescence in varied tissues, while also providing potential insights into the origin of urothelial cancer. We conclude that senescence of bladder uroepithelial cells plays a role in normal physiology, namely in their role as barrier cells, helping promote uroepithelial integrity and impermeability and maintaining the urine-blood barrier.

Keywords: D + Q; LUTS; aging; beneficial cellular senescence; bladder; blood‐urine barrier; lower urinary tract dysfunction; senolytics; tetraploidy‐induced senescence; umbrella cells.

FIGURE 1

Transcriptomic Characterization of Senescence and Aging in the Mouse Bladder. (a) Heat map of RT‐qPCR for senescent cell markers, SASP factors and immune cells in young (Y), middle aged (M) and old (O) female (left) and male (right) mouse bladders. N = 6–14 per age group for each sex. Relative gene expression calculated using geometric means shown. (b) RT‐qPCR data for p16 in female and male bladders over the lifespan. Kruskal–Wallis test with Dunn's correction for multiple comparisons. *p < 0.05 compared to Middle aged group. (c) Single cell sequencing data using pooled mouse bladders from different age groups to identify cells expressing senescent cell markers, SASP factors and genes whose expression changed significantly from middle to old age in our whole bladder RNA sequencing data. Top left, Cell type annotation of previous scRNA‐seq data performed on mouse bladders (GSE180128); Center right and Bottom, Expression of genes that were found to be differentially expressed in whole bladder RT‐qPCR data. (d) Whole bladder RNA sequencing data performed on young (Y, 2 months), middle‐aged (M, 10 months), and old (O, 26 months) mouse bladders. Displayed genes are those significant genes (adj. p‐value <0.05) with the highest |fold change| between middle‐aged and old bladders which have minimal change (|fold change| <0.2) between young and middle‐aged bladders. Expression (TPM) normalized to the middle aged group (n = 4–6 per age group). (e) Expression of genes found to be differentially expressed (|fold change| >1.5, adj p‐value <0.05) between old and middle samples in bulk RNA sequencing plotted in previous scRNA‐seq data. Example genes chosen based on intersection of significant bulk RNA seq gene set with marker genes identified from previous scRNA‐seq (GSE180128).

FIGURE 2

Identification of Senescent Cell Types in the Mouse Bladder. (a) Senescence Associated β‐galactosidase assay in female mouse bladder sections over the lifespan (Y = young (2–4 months), M = middle aged (10–12 months), O = old (20–26 months)). (b) Quantification of Senescence Associated β‐galactosidase assay in female mouse bladder sections over the lifespan. N = 3 mice/age group. Ordinary one‐way ANOVA, with Tukey test for multiple comparisons, *significant p < 0.05. (c) Representative image of P16 RNAscope in old mouse bladder surface urothelial cell. (d) Quantification of p16 RNAscope in middle aged and old mouse bladder urothelial nuclei. Unpaired t test with Welch's correction, two‐tailed, *significant p < 0.05, **p < 0.005, ***p < 0.0005. (e) Spatial transcriptomics data (Visium) (from GSE180128) with luminal urothelial cells isolated and expression of Cdkn2a in luminal urothelium compared between middle aged and old bladder section (n = 4, p‐value = 0.0349). (f) Telomere‐associated foci (TAFs) in a surface urothelial umbrella cell. Yellow arrowheads show colocalized telomere and γH2AX fluorescence, whereas white arrowheads show auto‐fluorescent lipofuscin droplets, the aging pigment, in cytoplasm of these same TAF positive cells. (g) IMARIS‐generated image identifying TAFs (yellow) based on overlap between surfaces created around telomere (green) and γH2AX (red) fluorescence. (h) Whole female mouse bladder section γH2AX staining (red), 20X stitched, showing high γH2AX expression in senescent surface luminal cells. (i) Quantification of TAF positive cells in urothelium in female mouse bladder sections over the lifespan N = 3 mice/age group. Ordinary one‐way ANOVA with Tukey's test for multiple comparisons, *significant p < 0.05.

FIGURE 3

Cyclin D1 staining in Bladder Uroepithelial (Umbrella) Cells. Immunofluorescence staining of Cyclin D1 protein in middle aged (10‐months) and old (26‐months) female mouse bladder sections. Negative controls did not receive primary antibody.

FIGURE 4

The Effect of D + Q Treatment on Senescence Markers in Old Mouse Bladders. (a) Schematic of senolytics drug treatment regimen used. Created with BioRender.com. (b) Senescence Associated β‐galactosidase assay in old (26‐months) female mouse bladders with Vehicle (V) or Dasatinib+Quercetin (D + Q) treatment. (c) Quantification of Senescence Associated β‐galactosidase assay in old (26‐months) female mouse bladders with V or D + Q treatment. N = 3 mice/treatment group. Unpaired two‐tailed t test, *significant p < 0.05. (d) Quantification of p16 RNAscope in old (26‐months) female mouse bladders with V or D + Q treatment. N = 3 mice/treatment group. Unpaired two‐tailed t test, *significant p < 0.05. (e) Quantification of TAF positive cells in urothelium in old (26‐months) female mouse bladders with V or D + Q treatment. N = 3 mice/treatment group. Unpaired two‐tailed t test, *significant p < 0.05. (f) Whole bladder RNA sequencing data showing most significantly upregulated genes from middle to old age whose expression was decreased by D + Q treatment, normalized to old. (n = 4–6 per group, middle aged (10 months), old (26 months) and D + Q‐treated old mouse bladders). Genes plotted are the top 20 significantly increased genes in the middle‐aged versus old analysis (adj. p‐value <0.05) that were also significantly decreased in the old versus D + Q analysis (adj. p‐value <0.05) after filtering to exclude technical effects (gavage/vehicle treatment). (g) Whole bladder RNA sequencing data showing most significantly downregulated genes from middle to old age whose expression was increased by D + Q treatment, normalized to old. (n = 4–6 per group, middle aged (10 months), old (26 months) and D + Q‐treated old mouse bladders). Genes plotted are the top 20 significantly decreased genes in the middle‐aged versus old analysis (adj. p‐value <0.05) that were also significantly increased in the old versus D + Q analysis (adj. p‐value <0.05) after filtering to exclude technical effects (gavage/vehicle treatment).

FIGURE 5

The Effect of D + Q Treatment on Aged Mouse Bladder Function. (a) Urodynamics tracing showing two sequential mouse voids and different parameters measured. NVC = nonvoiding contraction. IVI = intervoid interval. P_max: Maximum Bladder Pressure, Pbase: Baseline pressure, Pthresh: Threshold pressure, Pend: End of contraction. See Methods for other parameter explanation. (b) Maximum Bladder Pressure (P_max) difference between middle aged and old female mouse bladders detected by the cystometry protocol used for this study. No changes in other parameters were observed with aging. (n = 10 middle aged [M], 18 old [O]). Mean with SEM. Unpaired T test with Welch's correction, two‐tailed, *significant p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. (c) Voiding spot assays (VSA) carried out in middle aged and old female mice. (d) Quantification of VSA showed a significant difference in percentage of voided area in corners between middle aged and old female mice. (n = 10 middle aged [M], 31 old [O]). Mean with SEM. Unpaired nonparametric Mann–Whitney t test, two‐tailed, *significant p < 0.05. (e) Maximum Bladder Pressure (P_max) measured by cystometry did not change with D + Q treatment. No Tx = no treatment control, Water = water‐gavaged control. Mean with SEM. N = 8 no Tx, 18 water, 17 V and 16 D + Q. Ordinary one‐way ANOVA with Dunnett's test for multiple comparisons, compared to vehicle, *significant p < 0.05. (f) Percentage of voided area in corners measured by VSA did not change with D + Q treatment. No Tx = no treatment control, Water = water‐gavaged control. Mean with SEM. N = 11 no Tx, 22 water, 19 V and 25 D + Q. Ordinary one‐way ANOVA with Dunnett's test for multiple comparisons, compared to vehicle, *significant p < 0.05.

FIGURE 6

High‐Fat Diet and Senescent Cell Transplantation Induce Bladder Changes Similar to those Observed in Naturally Aged Mice. (a) Top panel: Schematic of High‐Fat Diet (HFD) or Normal Chow Diet (NCD) regimen used; Bottom panel: D + Q treatment regimen during HFD feeding regimen. Created with BioRender.com. (b) Maximum Bladder Pressure (P_max) difference between NCD‐ and HFD‐fed female mouse bladders detected by cystometry. No changes in other parameters were observed with HFD. (n = 8 NCD, 7 HFD). Mean with SEM. Unpaired t test, two‐tailed, *significant p < 0.05. (c) Voiding spot assays (VSA) carried out in NCD‐ and HFD‐fed female mice showed no difference in percentage of voided area in corners or any other VSA parameters studied. (n = 10 NCD, 9 HFD). Mean with SEM. Unpaired t test, two‐tailed, *significant p < 0.05. (d) Maximum Bladder Pressure (P_max) difference between HFD‐ and HFD followed by D + Q treatment (HFD + D + Q) in female mouse bladders measured by cystometry. No changes in other cystometric parameters were observed with HFD. (n = 7 NCD, 6 HFD). Mean with SEM. Unpaired t test, two‐tailed, *significant p < 0.05. (e) Schematic of senescent cell preparation and transplantation regimen used. 17‐months old female mice received PBS, Proliferating mouse ear fibroblasts (Prol.) or Senescent mouse ear fibroblasts (Sen.) intraperitoneally and bladder function assays carried out 2 months later. Created with BioRender.com. (f) Maximum Bladder Pressure (P_max) difference between female mouse bladders detected by the cystometry following IP injection of PBS, Proliferating or Senescent Mouse ear fibroblasts. No changes in other parameters were observed (n = 9 PBS, 10 Prol., 11 Sen.). Mean with SEM. Ordinary one‐way ANOVA, with Dunnett's test for multiple comparisons, compared to PBS, *significant p < 0.05. (g) Voiding spot assays (VSA) carried out following IP injection of PBS, Proliferating or Senescent Mouse ear fibroblasts showed no difference in percentage of voided area in corners or any other VSA parameters studied (n = 25 PBS, 23 Prol., 22 Sen.). Mean with SEM. Ordinary one‐way ANOVA, with Dunnett's test for multiple comparisons, *significant p < 0.05.