Introduction: E2 deficiency may induce fibrosis that leads to bladder dysfunction. We aimed at studying the effects of E2 on bladder function and assessing changes of bladder PDGFRa+ cells associated with inflammation and fibrosis.
Methods: C57BL/6 female mice were bilaterally ovariectomized (OVX) or sham surgery was performed at 7 weeks old and then E2 was applied subcutaneously in OVX mice for two weeks before experiment. Each protocol was done 1 , 3 and 7 months after OVX. In vivo cystometrogram (CMG), mice were restrained with specific holder under awake condition. qPCR, western blotting (WB) and immunohistochemistry (IHC) was carried out to investigate the relationship between E2 deficiency/replacement and fibrosis marker. To further explore this, bladder was divided into urothelium/suburothelial layer (UT) and detrusor smooth muscle layer (SM) and then cultured with control media, E2 (10 ng/ml) or TNFa (100 ng/ml) for 7 days. Cultured tissues were frozen at -80°C to study fibrosis marker.
Results: Bladder compliance was decreased in 1, 3 and 7 month OVX groups (20.3 ± 6.5, 10.2 ± 1.5 and 14.2 ± 2.7 µl/mmHg) compared to age-matched sham groups (35.1 ± 6.1, 24.6 ± 3.2 and 24.2 ± 4.5 µl/mmHg), while E2 recovered bladder compliance. Bladder capacity was reduced in 3 and 7 month OVX groups compared to sham , but was not fully recovered by E2. Intercontraction interval (ICI) was reduced in 3 and 7 month OVX groups (10.3 ± 1.8 and 10.7 ± 1.5 min) compared to age-matched sham groups (17.0 ± 1.1 and 17.0 ± 3.0 min) but was not rescued by E2 treatment. Unexpectedly, E2 markedly increased the number of non-voiding contractions (NVCs). Estrogen receptor (ERa) was dominantly immunoreactive against detrusor, however bladder interstitial cells were immunopositive upon ERß. Colocalizations of PDGFRa+ and ERß positive interstitial cells were observed both in UT and SM. Expression of PDGFRa and collagen 1a1 was upregulated in 3 month OVX group compared to sham, while E2 replacement further increased the expression of PDGFRa and fibrosis markers compared to age-matched sham or OVX groups. In qPCR of cultured bladder tissue, TNFa dramatically upregulated the expression of Acta2, TIMP2, Tgfß, Col1a1, Col1a2, cadherin and IL6 in UT but had marginal effects on SM.
Conclusions: Urothelium/suburothelial layer may be common site for the initiation of fibrosis. Inflammation or E2 deficiency/replacement may cause fibrosis through the activation of PDGFRa+ cells. These novel findings might point to a novel approach toward the treatment of bladder fibrosis.
Source of Funding: Supported by NIDDK, RO1 DK098388
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