Underactive bladder (UAB), characterized by a complex set of symptoms with few treatment options, can significantly reduce the quality of life of affected people. UAB is characterized by hyperplasia and fibrosis of the bladder wall as well as decreased bladder compliance. Pirfenidone is a powerful anti-fibrotic agent that inhibits the progression of fibrosis in people with idiopathic pulmonary fibrosis. In the current study, we evaluated the efficacy of pirfenidone in the treatment of bladder fibrosis in a UAB rat model. UAB was induced by crushing damage to nerve bundles in the major pelvic ganglion. Forty-two days after surgery, 1 mL distilled water containing pirfenidone (100, 300, or 500 mg/kg) was orally administered once every 2 days for a total of 10 times for 20 days to the rats in the pirfenidone-treated groups. Crushing damage to the nerve bundles caused voiding dysfunction, resulting in increased bladder weight and the level of fibrous related factors in the bladder, leading to UAB symptoms. Pirfenidone treatment improved urinary function, increased bladder weight and suppressed the expression of fibrosis factors. The results of this experiment suggest that pirfenidone can be used to ameliorate difficult-to-treat urological conditions such as bladder fibrosis. Therefore, pirfenidone treatment can be considered an option to improve voiding function in patient with incurable UAB.

OBJECTIVE: Diabetic bladder fibrosis is a common comorbidity. Altered expression of some long non-coding RNAs (LncRNAs) has been associated with bladder fibrosis. LncRNA H19 has been reported to regulate bladder cancer through miR-29b. However, the action mechanism of LncRNA H19 in bladder fibrosis is unclear.
METHODS: In vitro, human bladder smooth muscle cells (HBSMCs) were cultured with transforming growth factor-β1 (TGF-β1) for 48 h to construct cell model of bladder fibrosis. HBSMCs were then transfected with si-LncRNA H19, si-NC, miR-29b-mimic, mimic-NC, or miR-29b-inhibitor. In vivo, Sprague-Dawley (SD) rats were given a high-sucrose-high-fat (HSHF) diet for 4 weeks and injected with streptozotocin (STZ, 50 mg/kg) to induce bladder fibrosis model in diabetic rats, followed by injection of lentiviral particles knocking down LncRNA H19 expression, empty vector, or miR-29b-inhibitor, respectively.
RESULTS: LncRNA H19 was up-regulated in TGF-β1-induced HBSMC fibrosis and STZ-induced diabetic rat bladder fibrosis, whereas miR-29b was down-regulated. si-LncRNA H19 reduced blood glucose levels and improved histopathological damage of bladder tissue in rats. In addition, si-LncRNA H19 or miR-29b-mimic increased the expression of E-cadherin, but decreased the expression of N-cadherin, vimentin, fibronectin (FN) in bladder tissues, and HBSMCs. si-LncRNA H19 reduced TGF-β1/p-drosophila mothers against decapentaplegic 3 (Smad3) protein in HBSMCs and in rat bladder tissues, while miR-29b-inhibitor reversed the effect of si-LncRNA H19.
CONCLUSIONS: This study indicated that LncRNA H19 may inhibit bladder fibrosis in diabetic rats by targeting miR-29b via the TGF-β1/Smad3 signalling pathway.

Neurogenic bladder (NB) following suprasacral spinal cord injury (SSCI) is an interstitial disease with the structural remodeling of bladder tissue and matrix over-deposition. Circular RNAs (circRNAs) are involved in fibrotic disease development through their post-transcriptional regulatory functions. This study aimed to use transcriptome high-throughput sequencing to investigate the process of NB and bladder fibrosis after SSCI.
Spinal cord transection at the T10-T11 level was used to construct the SSCI model in rats (10-week-old female Wistar rats, weighing 200 ± 20 g). The bladders were collected without (sham group) and with (SSCI 1-3 groups) NB status. Morphological examination was conducted to assess the extent of bladder fibrosis. Additionally, RNA sequencing was utilized to determine mRNAs and circRNAs expression patterns. The dynamic changes of differentially expressed mRNAs (DEMs) and circRNAs (DECs) in different periods of SSCI were further analyzed.
Bladder weight, smooth muscle cell hypertrophy, and extracellular matrix gradually increased after SSCI. Compared with the sham group, 3,255 DEMs and 1,339 DECs, 3,449 DEMs and 1,324 DECs, 884 DEMs, and 1,151 DECs were detected in the SSCI 1-3 groups, respectively. Specifically, circRNA3621, circRNA0617, circRNA0586, and circRNA4426 were significant DECs common to SSCI 1-3 groups compared with the sham group. Moreover, Gene Ontology (GO) enrichment suggested that inflammatory and chronic inflammatory responses were the key events in NB progression following SSCI. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment associated with the \"Chemokine signaling pathway\", the \"IL-17 signaling pathway\", and the \"TGF-beta signaling pathway\" suggests their potential involvement in regulating biological processes. The circRNA-miRNA-mRNA interaction networks of DECs revealed rno-circ-2239 (micu2) as the largest node, indicating that the rno-circ-2239-miRNA-mRNA-mediated network may play a critical role in the pathogenesis of SSCI-induced NB.
This study offers a comprehensive outlook on the possible roles of DEMs and DECs in bladder fibrosis and NB progression following SSCI. These findings have the potential to serve as novel biomarkers and therapeutic targets.

The activation of the transient receptor potential ankyrin 1 (TRPA1) channel has anti-fibrotic effects in the lung and intestine. Suburothelial myofibroblasts (subu-MyoFBs), a specialized subset of fibroblasts in the bladder, are known to express TRPA1. However, the role of the TRPA1 in the development of bladder fibrosis remains elusive. In this study, we use the transforming growth factor-β1 (TGF-β1) to induce fibrotic changes in subu-MyoFBs and assess the consequences of TRPA1 activation utilizing RT-qPCR, western blotting, and immunocytochemistry. TGF-β1 stimulation increased α-SMA, collagen type I alpha 1 chain(col1A1), collagen type III (col III), and fibronectin expression, while simultaneously suppressing TRPA1 in cultured human subu-MyoFBs. The activation of TRPA1, with its specific agonist allylisothiocyanate (AITC), inhibited TGF-β1-induced fibrotic changes, and part of these inhibition effects could be reversed by the TRPA1 antagonist, HC030031, or by reducing TRPA1 expression via RNA interference. Furthermore, AITC reduced spinal cord injury-induced fibrotic bladder changes in a rat model. The increased expression of TGF-β1, α-SMA, col1A1 and col III, and fibronectin, and the downregulation of TRPA1, were also detected in the mucosa of fibrotic human bladders. These findings suggest that TRPA1 plays a pivotal role in bladder fibrosis, and the negative cross talk between TRPA1 and TGF-β1 signaling may represent one of the mechanisms underlying fibrotic bladder lesions.

Lower urinary tract symptoms (LUTS) refer to various urological diseases, and incomplete bladder emptying is common among affected patients. The etiology of LUTS is largely unknown, and investigations of LUTS suggest that bladder fibrosis contributes to pathogenesis of LUTS. MicroRNAs (miRNAs) are short (∼22 nucleotides), non-coding RNAs that repress target gene expression by a combination of mRNA degradation and translation inhibition. The miR-29 family is best known for its anti-fibrotic role in various organs. miR-29 was decreased in bladders of patients with outlet obstruction and a rat model of bladder outlet obstruction, suggesting that miR-29 may contribute to impaired bladder function subsequent to tissue fibrosis. We characterized bladder function in male mice lacking expression of Mir29a and Mir29b-1 (miR-29a/b1). Lack of miR-29a/b1 resulted in severe urinary retention, increased voiding duration and reduced flow rate, and these mice failed to void or voided irregularly during anesthetized cytometry. Collagens and elastin were increased in bladders of mice lacking miR-29a/b1. These findings reveal an important role for miR-29 in bladder homeostasis and suggest the therapeutic potential of miR-29 to improve symptoms in patients with LUTS.

BACKGROUND: In the last twenty-five years, Onabotulinum Toxin A (BTX-A) has gained increasing popularity for neurogenic lower urinary tract dysfunction (NLUTD) treatment. To maintain its efficacy, repeated BTX-A intradetrusor injections are required over time, with unknown effects on the bladder wall in children. The aim of this paper is to report long-term effects on the bladder wall in children treated with BTX-A.
METHODS: Children with NLUTD not responsive to anticholinergics were treated with BTX-A, according to our protocol, with bladder wall control using endoscopic cold-cup biopsy. Specimens were evaluated considering edema, chronic inflammation, and fibrosis.
RESULTS: Of the 230 patients treated from 1997 to 2022, we considered only specimens obtained in patients who had received ≥5 treatments (36 children), considered as the threshold to evaluate clinical effectiveness on long-term treatment with BTX-A. Most of them had congenital NLUTD (25 patients) and detrusor overactivity (27 patients). In all, increased edema and chronic inflammation with reduced fibrosis over time was reported; these data were not statistically significant. No difference was observed between patients with congenital and acquired diseases.
CONCLUSIONS: Repeated intradetrusor BTX-A injections are not related to significant histological alterations in children, similarly with adults, and repeated injections could be considered safe.

Lower urinary tract symptoms (LUTS) refer to various urological diseases, and incomplete bladder emptying is common among affected patients. Etiology of LUTS is largely unknown, and investigations of LUTS suggest that bladder fibrosis contributes to pathogenesis of LUTS. MicroRNAs (miRNAs) are short (∼22 nucleotides), non-coding RNAs that repress target gene expression by a combination of mRNA degradation and translation inhibition. The miR-29 family is best known for its anti-fibrotic role in various organs. miR-29 was decreased in bladders of patients with outlet obstruction and a rat model of bladder outlet obstruction, suggesting that miR-29 may contribute to impaired bladder function subsequent to tissue fibrosis. We characterized bladder function in male mice lacking expression of MIR29A and MIR29B1 (miR-29a/b1). Lack of miR-29a/b1 resulted in severe urinary retention, increased voiding duration with reduced flow rate, and these mice failed to void or voided irregularly during anesthetized cytometry. Collagens and elastin were increased in bladders of mice lacking miR-29a/b1. These findings reveal an important role of mir-29 in bladder homeostasis and suggest therapeutic potential of miR-29 to improve symptoms in patients with LUTS.

OBJECTIVE: The overactive bladder syndrome (OAB) is a bothersome condition that affects up to 33% of the population. In up to 69% of the cases, the underlying condition is an overactive detrusor (DO). Treatment options rely on behavioral changes, medical treatment, neuromodulation, and invasive treatment, such as injecting botulinum toxin (BoNT) in the detrusor or augmentation cystoplasty. The aim of this study was to evaluate, by morphological assessment on cold-cup biopsies of the bladder, the effect of botulinum toxin injections on the bladder wall, focusing on the histological structure and signs of inflammation and fibrosis.
METHODS: We evaluated consecutive patients with DO that received BoNT intradetrusor injections. We analyzed inflammation and fibrosis in 36 patients, divided into two groups based on their history of BoNT treatment. Our patients underwent at least one round of injections and specimens were compared individually, before and after each injection.
RESULTS: A decrease in inflammation was found in 26.3% of the cases, a reactive increase in 31.5%, and no change in 42.1%. No de novo or increase in preexisting fibrosis was found. In some cases, fibrosis diminished after a second round of BoNT.
CONCLUSIONS: In most cases, BoNT intradetrusor injections in DO patients showed no effect on bladder wall inflammation and actually improved the inflammatory condition of the muscle in a significant number of samples.

Bladder outlet obstruction (BOO) is a prevalent condition arising from urethral stricture, posterior urethral valves, and benign prostatic hyperplasia. Long-term obstruction can lead to bladder remodeling, which is characterized by inflammatory cell infiltration, detrusor hypertrophy, and fibrosis. Until now, there are no efficacious therapeutic options for BOO-induced remodeling. Tetrahedral framework nucleic acids (tFNAs) are a type of novel 3D DNA nanomaterials that possess excellent antifibrotic effects. Here, to determine the treatment effects of tFNAs on BOO-induced remodeling is aimed. Four single-strand DNAs are self-assembled to form tetrahedral framework DNA nanostructures, and the antifibrotic effects of tFNAs are investigated in an in vivo BOO animal model and an in vitro transforming growth factor beta1 (TGF-β1)-induced fibrosis model. The results demonstrated that tFNAs could ameliorate BOO-induced bladder fibrosis and dysfunction by inhibiting M2 macrophage polarization and the macrophage-myofibroblast transition (MMT) process. Furthermore, tFNAs regulate M2 polarization and the MMT process by deactivating the signal transducer and activator of transcription (Stat) and TGF-β1/small mothers against decapentaplegic (Smad) pathways, respectively. This is the first study to reveal that tFNAs might be a promising nanomaterial for the treatment of BOO-induced remodeling.

Fibrosis is a chronic inflammation process with excess extracellular matrix (ECM) deposition that cannot be reversed. Patients suffer from bladder dysfunction caused by bladder fibrosis. Moreover, the interactive mechanisms between ECM and bladder fibrosis are still obscure. Hence, we assessed the pivotal effect of Yes-associated protein (YAP) on the proliferation of bladder smooth muscle in fibrosis process. We identified that stiff ECM increased the expression and translocation of YAP in the nucleus of human bladder smooth muscle cell (hBdSMC). Sequencings and proteomics revealed that YAP bound to Smad3 and promoted the proliferation of hBdSMC via MAPK/ERK signaling pathway in stiff ECM. Moreover, CUT and TAG sequencing and dual-luciferase assays demonstrated that Smad3 inhibited the transcription of JUN. The YAP inhibitor CA3 was used in a partial bladder outlet obstruction (pBOO) rat model. The results showed that CA3 attenuated bladder smooth muscle proliferation. Collectively, YAP binding with Smad3 in the nucleus inhibited the transcription of JUN, and promoted the proliferation of bladder smooth muscle through the MAPK/ERK signaling pathway. The current study identified a novel mechanism of mechanical force induced bladder fibrosis that provided insights in YAP-associated organ fibrosis.