Exosomes from human urine-derived stem cells carry NRF1 to alleviate bladder fibrosis via regulating miR-301b-3p/TGFßR1 pathway.

Bladder outlet obstruction (BOO) is a common disease that always make the bladder develops from inflammation to fibrosis. This study was to investigate the effect of exosomes from human urine-derived stem cells (hUSCs) on bladder fibrosis after BOO and the underlying mechanism. The BOO mouse model was established by inserting a transurethral catheter, ligation of periurethral wire, and removal of the catheter. Mouse primary bladder smooth muscle cells (BSMCs) were isolated and treated with TGFß1 to mimic the bladder fibrosis model in vitro. Exosomes from hUSCs (hUSC-Exos) were injected into the bladder of BOO mice and added into the culture of TGFß1-induced BSMCs. The associated factors in mouse bladder tissues and BSMCs were detected. It was confirmed that the treatment of hUSC-Exos alleviated mouse bladder fibrosis and down-regulated fibrotic markers (a-SMA and collagen III) in bladder tissues and TGFß1-induced BSMCs. Overexpression of NRF1 in hUSC-Exos further improved the effects of hUSC-Exos on bladder fibrosis both in vivo and in vitro. TGFßR1 was a target of NRF1 and miR-301b-3p, and miR-301b-3p was a target of NRF1. It was next characterized that hUSC-Exos carried NRF1 to up-regulate miR-301B-3p, thereby reducing TGFßR1level. Our results illustrated that hUSC-Exos carried NRF1 to alleviate bladder fibrosis through regulating miR-301b-3p/TGFßR1 pathway.

Activation of TRPA1 in Bladder Suburothelial Myofibroblasts Counteracts TGF-ß1-Induced Fibrotic Changes.

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-ß1 (TGF-ß1) to induce fibrotic changes in subu-MyoFBs and assess the consequences of TRPA1 activation utilizing RT-qPCR, western blotting, and immunocytochemistry. TGF-ß1 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-ß1-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-ß1, 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-ß1 signaling may represent one of the mechanisms underlying fibrotic bladder lesions.

Transcriptomic analysis of bladder tissue in a rat model of ketamine-induced bladder fibrosis.

INTRODUCTION: Ketamine-induced cystitis (KIC) is a disease caused by ketamine that can cause lower urinary tract symptoms (LUTS). Its end-stage is bladder contracture, which is related to bladder fibrosis and poses a serious burden to patient lives. METHODS: We established a KIC model in female Sprague Dawley rats and verified bladder fibrosis in the model by Masson trichrome staining and western blot analysis. The bladders of the rats from the ketamine and control groups were used to perform transcriptome analysis. In particular, association analysis with metabolomics was also used to determine the potential mechanisms of ketamine-induced bladder fibrosis. RESULTS: A total of 685 differentially expressed messenger RNAs, 71 differentially expressed long noncoding RNAs, 23 differentially expressed microRNAs, and 68 differentially expressed circular RNAs were identified. We found that ribosome, Wnt signaling, vascular endothelial growth factor signaling, cytoskeleton organization, and cytoskeletal protein binding may be potential pathways in ketamine-induced bladder fibrosis as identified by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. In addition, the mitogen-activated protein kinase pathway appeared to be closely related to the development of ketamine-induced bladder fibrosis according to association analysis. CONCLUSIONS: In this study, using transcriptomic and correlation analyses of metabolomics, we identified pathways that may be potential targets for the prevention and treatment of ketamine-induced bladder fibrosis.

Partial inhibition of activin receptor-like kinase 4 alleviates bladder fibrosis caused by bladder outlet obstruction.

The bladder undergoes profound structural alterations after bladder outlet obstruction (BOO), characterized by hypertrophy of the bladder wall and accumulation of extracellular matrix (ECM). Transforming growth factor-ß (TGF-ß) has been found to promote fibrosis of the bladder induced by partial bladder outlet obstruction (pBOO). Activin receptor-like kinase 4 (ALK4) is a downstream receptor of the TGF-ß superfamily. However, the role of the ALK4-Smad2/3 pathway in the pathogenesis of bladder fibrosis caused by pBOO remains unknown. This study focused on learning the role of ALK4 in the process of bladder fibrosis caused by pBOO. The pBOO mice models showed that ALK4 expression was found to upregulate in the wild-type bladder 6 weeks after pBOO compared to control group. Then, mice with heterozygous knockout of the ALK4 gene (ALK4+/-) were generated. Histological analysis and Western blot (WB) results showed significant suppression of collagen expression in the bladders of ALK4+/- mice after pBOO compared with WT mice. WB also showed that ALK4+/- mice demonstrated significant suppression of phosphorylated Smad2/3 (p-Smad2/3) expression in the bladder 6 weeks after pBOO but not of phosphorylated extracellular signal-regulated kinase, c-Jun N-terminal kinase or protein 38 (p-ERK, p-JNK, p-P38) expression. This effect might have occurred through partial inactivation of the Smad2/3 signaling pathway. In vitro, ALK4 overexpression promoted collagen production in cultured BSMCs and activated the Smad2/3 signaling pathway. Taken together, our results demonstrated that ALK4 insufficiency alleviated bladder fibrosis in a mouse model of pBOO partly by suppressing Smad2/3 activity.

Antifibrosis treatment by inhibition of VEGF, FGF, and PDGF receptors improves bladder wall remodeling and detrusor overactivity in association with modulation of C-fiber afferent activity in mice with spinal cord injury.

AIMS: Spinal cord injury (SCI) above the sacral level causes bladder dysfunction and remodeling with fibrosis. This study examined the antifibrotic effects using nintedanib, an inhibitor of vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor receptors, on detrusor overactivity (DO) and bladder fibrosis, as well as the modulation mechanisms of C-fiber afferent pathways. METHODS: Thirty female C57BL/6 mice were divided into group A (spinal intact), group B (SCI with vehicle), and group C (SCI with nintedanib). At 2 weeks after SCI, vehicle or 50 mg/kg nintedanib was administered subcutaneously for 2 weeks. Then, cystometry was conducted, followed by RT-PCR measurements of fibrosis-related molecules, muscarinic, ß-adrenergic, TRP and purinergic receptors in the bladder or L6-S1 dorsal root ganglia (DRG). Trichrome stain and Western blot analysis of transforming growth factor-beta and fibronectin were performed in the bladder. TRPV1 expression in L6 DRG was measured by immunohistochemistry. RESULTS: In cystometry, intercontraction intervals, nonvoiding contractions, voided volume, and voiding efficiency were significantly improved in group C versus group B. RT-PCR, Western blotting, and trichrome staining revealed the fibrotic changes in the bladder of group B, which was improved in group C. Increased messenger RNA levels of TRPV1, TRPA1, P2X2 , and P2X3 in DRG of group B were significantly decreased in group C. TRPV1 immunoreactivity in DRG was increased in group B, but decreased in group C. CONCLUSIONS: Nintedanib improves storage and voiding dysfunctions and bladder fibrosis in SCI mice. Also, nintedanib-induced improvement of DO is associated with reduced expression of C-fiber afferent markers, suggesting the modulation of bladder C-fiber afferent activity.

Losartan prevents bladder fibrosis and protects renal function in rat with neurogenic paralysis bladder.

AIMS: To investigate the effect of losartan on preventing bladder fibrosis and protecting renal function in rats with neurogenic paralysis bladder (NPB). MATERIALS AND METHODS: Rats were assigned to the transecting spinal nerves group (TSNG), transecting spinal nerves + losartan group (LSTG), and control group (CG). On Day 32 postsurgery, bladder capacity (BC), bladder compliance (ΔC), bladder leakage pressure (Pves.leak ) of TSNG and LSTG while BC, ΔC, and bladder threshold pressure (Pves.thre ) of CG were measured by cystometry in each cohort. Renal function and the expression quantity of Angiotensin Ⅱ (Ang II) in blood were detected, in addition Ang II, Ang II Type 1 receptor (AT1), transformation growth factor ß1 (TGFß1), Collagen Ⅲ, and collagen fibrin in the bladder tissue were detected too. RESULTS: ΔC in TSNG and LSTG decreased significantly compared to the CG. Pves.leak in TSNG and LSTG were significantly higher than Pves.thre in CG. Renal function of both TSNG and LSTG decreased significantly compared with the CG, but renal function in LSTG was better than in TSNG. Ang Ⅱ in blood and bladder tissue in TSNG and LSTG increased significantly compared with CG. AT1 was expressed in the bladder tissue of all rats. The TGFß1, Collagen Ⅲ, and collagen fibrin expression level increased significantly in TSNG compared with LSTG and CG, while these levels were not significantly different between CG and LSTG. CONCLUSION: Losartan might prevent NPB fibrosis by stopping the upregulated signaling of Ang II/AT1/TGFß1 and consequently may reduce kidney damage from occurring.

MicroRNA-495 alleviates ulcerative interstitial cystitis via inactivating the JAK-STAT signaling pathway by inhibiting JAK3.

INTRODUCTION AND HYPOTHESIS: As a notable chronic disorder, the incidence of interstitial cystitis (IC) has been documented to have increased among the female population with activity in microRNA-495 (miR-495) implicated in this disease. The current study was aimed at elucidating the effects associated with miR-495 on the inflammatory response and bladder fibrosis in rats with ulcerative IC via the JAK-STAT pathway by targeting JAK3. METHODS: Ulcerative IC rat models were established. The targeting relationship between JAK3 and miR-495 was evaluated using luciferase reporter assay. After gain- or loss-of-function assays, mast-cell infiltration was assessed using toluidine blue staining, bladder fibrosis using Masson staining, and NO content using nitrate reductase method. JAK3 protein expression was detected by immunohistochemistry, JAK3, STAT1, STAT3, TGFß-1, Col-I, Col-III, JAK1, JAK2, p-STAT1, and p-STAT3 expression by RT-qPCR and Western blot analysis, and serum IL-6, IL-8, IL-10, IL-17, and TNF-α levels in rats by ELISA. RESULTS: Following transfection of overexpressed miR-495 or siRNA-JAK3, a diminished degree of mast-cell infiltration, number of mast cells, bladder fibrosis, NO content, JAK3-positive expression, mRNA expression of JAK3, STAT1, STAT3, TGFß-1, Col-I, Col-III, protein expression of JAK1, JAK2, JAK3, p-STAT1, p-STAT3, and expression of IL-6, IL-8, IL-10, IL-17, and TNF-α were identified. CONCLUSIONS: Collectively, our key findings provide evidence supporting the notion that the overexpression of miR-495 ameliorates inflammatory response and bladder fibrosis in ulcerative IC rat models via inactivation of the JAK-STAT signaling pathway by inhibiting JAK3.

Long-term tadalafil administration can prevent functional and structural changes of the urinary bladder in male rats with partial bladder outlet obstruction.

AIMS: There have been few reports on whether long-term oral phosphodiesterase 5 inhibitor administration can ameliorate bladder changes due to bladder outlet obstruction (BOO). Therefore, we clarified the chronological changes of the bladder using male BOO rats and evaluated the effects of tadalafil on these changes. METHODS: Eight-week-old male Sprague-Dawley rats were used. BOO was created by placing a polyethylene catheter around the urethra. Then, the rats were orally treated with a vehicle, or tadalafil 2 or 10 mg/kg until each evaluation period. Cystometric measurements were performed and the degree of fibrosis in the smooth muscle layer was evaluated at 2, 4, and 16 weeks. RESULTS: In BOO rats, a significant increase in the number of non-voiding contractions (NVCs) and a shortened intercontraction interval (ICI) were observed in the earlier phase (2 and 4 weeks) compared to Sham rats. In the chronic phase (16 weeks), markedly increased residual urine volume and an extended ICI were observed accompanied by enhanced smooth muscle fibrosis. These results indicated that the bladder in BOO rats represented the overactive phenotype in the earlier phase and changed into the underactive phenotype in the chronic phase. Even in Sham rats, an increased number of NVCs and enhanced fibrosis were observed with time. Tadalafil administration significantly prevented these bladder changes in both BOO and Sham rats. CONCLUSIONS: Long-term oral administration of tadalafil can prevent functional and histological changes in the BOO rat bladder. This agent is also effective for the bladder functional change even in non-obstructed rats.

MicroRNA-219c-5p regulates bladder fibrosis by targeting FN1.

BACKGROUND: We found that the bladders of multiple sclerosis mice were significantly fibrotic. This study aimed to investigate the relationship between fibronectin 1 (FN1) and bladder fibrosis, as well as the microRNAs involved in FN1 regulation. METHODS: The degree of bladder smooth muscle fibrosis was observed by immunohistochemistry. In addition, we used quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting to determine FN1 expression in bladders with different grades of fibrosis. Bioinformatics analysis revealed that miR-199a-3p, miR-219c-5p and miR-3572-3p could inhibit FN1 synthesis. Therefore, miR-199a-3p, miR-219c-5p and miR-3572-3p were overexpressed or knocked down in bladder smooth muscle cells (BSMCs), and the respective transfection and FN1 knockdown efficiencies were detected by RT-qPCR. Only miR-219c-5p overexpression and knockdown produced the expected results. A dual luciferase reporter assay was used to determine the targeting relationship between miR-219c-5p and FN1. Flow cytometry and Cell Counting Kit 8 (CCK8) experiments confirmed that miR-219c-5p reduced FN1 expression and affected the biological activity of smooth muscle cells. Agomir and anagomir of miR-219c-5p were transfected in vivo to observe the change of bladder fibrosis in mice. RESULTS: With increasing bladder fibrosis, FN1 expression increased, while miR-199a-3p, miR-219c-5p, and miR-3572-3p expression levels decreased. The RT-qPCR results after transfection showed that only miR-219c-5p could regulate FN1. Indeed, the dual luciferase reporter assay results indicated that miR-219c-5p targeted FN1 directly. CCK8 and cell cycle assays showed that miR-219c-5p overexpression inhibited BSMC proliferation, while miR-219c-5p knockdown promoted BSMC proliferation. An apoptosis assay showed that miR-219c-5p overexpression promoted apoptosis, while miR-219c-5p knockdown inhibited BSMC apoptosis. The agomir and anagomir transfected with miR-219c-5p in vivo found that the bladder fibrosis of the mice in the agomir group was reduced, and the anagomir group was worse. CONCLUSIONS: Our findings indicate that FN1 up-regulation and miR-219c-5p down-regulation play an important role in the development of bladder fibrosis, and miR-219c-5p participates in bladder fibrosis by regulating FN1 expression. Thus, a novel antifibrotic function of miR-219c-5p is proposed, which may represent a potential target for the diagnosis and treatment of bladder fibrosis.

MicroRNA-101 protects bladder of BOO from hypoxia-induced fibrosis by attenuating TGF-ß-smad2/3 signaling.

Bladder outlet obstruction is a common disease, which always evokes urinary bladder wall remodeling significantly. It has been suggested that bladder outlet obstruction can make the bladder progression from inflammation to fibrosis, and hypoxia may play a vital role. It has been found the expression of microRNA-101 varied in bladder after BOO. But what role microRNA-101 and hypoxia play in bladder is not well known. This study is to investigate the mechanism of microRNA-101 and hypoxia in fibrosis of bladder after BOO. We found the expression of microRNA-101 and hif-1α increased in bladder after BOO. Hypoxia could promote the expression of extracellular matrix subtypes and microRNA-101 in BSMCs. When microRNA-101b was translated into BSMCs, the smad2/3 signaling pathway was found to repress. Dual luciferase reporter detected that microRNA-101b attenuated the TGF-ß signaling pathway by inhibiting the expression of TGFßR1. Then, we conclude microRNA-101b is induced by hypoxia and represses fibrosis of BSMCs by inhibiting the expression of TGFßR1 through TGF-ß signaling pathway, and it may be an anti-fibrotic miRNA for therapy. © 2018 IUBMB Life, 71(1):235-243, 2019.

Exploring the protective effect and mechanism of icariside II on the bladder in a rat model of radiation cystitis based on transcriptome sequencing.

PURPOSE: Radiation cystitis (RC) is a complex and common complication after radiotherapy for pelvic cancer. Icariside II (ICAII) is a flavonoid compound extracted from Epimedium, a traditional Chinese medicine, with various pharmacological activities. The aim of the present study was to investigate the cysto-protective effects of ICAII in RC rats and its possible mechanisms. MATERIALS AND METHODS: A rat model of induced radiation cystitis using pelvic X-ray irradiation was used, and bladder function was assessed by bladder volume and bladder leakage point pressure (LPP) after ICAII treatment. HE and Masson stains were used to assess the histopathological changes in the bladder. IL-6, TNF-α, IL-10, IL-4 and IL-1ß were measured by ELISA to assess the level of inflammation. The gene-level changes in ICAII-treated RC were observed by transcriptome sequencing, and then the potential targets of action and biological mechanisms were explored by PPI, GO and KEGG enrichment analysis of the differentially expressed genes. Finally, the predicted targets of action were experimentally validated using immunohistochemistry, RT-qPCR, molecular docking and CETSA. RESULTS: ICAII significantly increased bladder volume and the LPP, ameliorated pathological damage to bladder tissues, decreased the levels of IL-6, TNF-α, and IL-1ß, and increased the levels of IL-10 and IL-4 in radiation-injured rats. A total of 90 differentially expressed genes were obtained by transcriptome sequencing, and PPI analysis identified H3F3C, ISG15, SPP1, and LCN2 as possible potential targets of action. GO and KEGG analyses revealed that these differentially expressed genes were mainly enriched in the pathways metabolism of xenobiotics by cytochrome P450, arachidonic acid metabolism, Staphylococcus aureus infection and chemical carcinogenesis - reactive oxygen species. Experimental validation showed that ICAII could significantly increase the expression of H3F3C and ISG15 and inhibit the expression of SPP1 and LCN2. ICAII binds well to H3F3C, ISG15, SPP1 and LCN2, with the best binding ability to H3F3C. Furthermore, ICAII inhibited the protein degradation of H3F3C in bladder epithelial cells. CONCLUSIONS: ICAII may alleviate the bladder inflammatory response and inhibit the fibrosis process of bladder tissues through the regulation of H3F3C, ISG15, SPP1, and LCN2 targets and has a protective effect on the bladder of radioinjured rats. In particular, H3F3C may be one of the most promising therapeutic targets.

Pirfenidone improves voiding function by suppressing bladder fibrosis in underactive bladder rats.

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.

Long non-coding RNA H19 mediates the miR-29b/transforming growth factor-ß1/Drosophila mothers against decapentaplegic 3 signalling pathway to promote bladder fibrosis in diabetic rats.

PURPOSE: 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-ß1 (TGF-ß1) 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-ß1-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-ß1/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. CONCLUSION: This study indicated that LncRNA H19 may inhibit bladder fibrosis in diabetic rats by targeting miR-29b via the TGF-ß1/Smad3 signalling pathway.

Bladder Wall Structure Alterations in Patients Treated With Botulinum Toxin for Detrusor Overactivity - A Morphological Study.

BACKGROUND/AIM: 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. PATIENTS AND 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. CONCLUSION: 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.

Lack of expression of miR-29a/b1 impairs bladder function in male mice.

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.

Antifibrotic Effects of Tetrahedral Framework Nucleic Acids by Inhibiting Macrophage Polarization and Macrophage-Myofibroblast Transition in Bladder Remodeling.

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-ß1)-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-ß1/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.

Placenta accreta spectrum with severe morbidity: fibrosis associated with cervical-trigonal invasion.

OBJECTIVE: Describe the clinical-surgical results of patients with PAS in the low-posterior cervical-trigonal space associated with fibrosis (PAS type 4) compared with PAS types in other locations (Types 1, upper bladder, 2 in upper parametrium) and in particular with PAS type 3, corresponding to dissectible cervical-trigonal invasion. The clinical-surgical results of using a standard hysterectomy were analyzed with a modified subtotal hysterectomy (MSTH) in patients with PAS type 4. MATERIAL AND METHODS: A descriptive, retrospective, multicenter study included 337 patients of PAS; thirty-two corresponding to PAS type 4, from three PAS reference hospitals, CEMIC, Buenos Aires, Argentina, Fundación Valle de Lili, Cali, Colombia, and Dr. Soetomo General Hospital, Surabaya, Indonesia, between January 2015 and December 2020. PAS was diagnosed by abdominal and transvaginal ultrasound and topographically characterized by ultrafast T2 weighted MRI. In persistent macroscopic hematuria after MSTH, the surgeon performs an intentional cystotomy and uses a square compression suture to achieve the hemostasis inside the bladder wall.According to a PAS topographical classification, the patients with low-vesical cervical involvement compared with PAS located in relation with the upper blader (type1), upper parametrium (type 2 upper), and also with PAS situated in the lower vesical-trigon space (type 3). PAS 3 and 4 are located in identical area, but in type 3, group A, the vesicouterine space was dissectible, and in type 4, group B, significant fibrosis made surgical dissection extremely challenging. Furthermore, group B was divided into patients treated with total hysterectomy (HT) and those treated with a modified subtotal hysterectomy (MSTH). The surgical requirements to perform an MSHT included the availability of proximal vascular control at the aortic level (internal manual aortic compression, aortic endovascular balloon, aortic loop, or aortic cross-clamping). Then surgeon performed an upper segmental hysterotomy, avoiding the abnormal placenta invasion area; after that, the fetus was delivered, and the umbilical cord was ligated.After uterine exteriorization, the surgeon applies a continuous circular suture with number 2 polyglactin 910, taking some portions of the myometrium -to avoid unintentional slipping- around the lower uterine segment and a 3-4 cm proximal to the abnormal adhesion of the placenta. After tightening hard the circular suture, the uterine segment was circumferentially cut, three centimeters proximal to the circular hemostatic sutures. Next, the surgery follows the upper steps of conventional hysterectomy without changes. Additionally, the histological presence of fibrosis was examined in all samples. RESULTS: Modified subtotal hysterectomy in patients with PAS type 4 (cervical-trigonal fibrosis) resulted in a significant clínico-surgical improvement over total hysterectomy. The median operative time and intraoperative bleeding were 140 min (IQR 90--240) and 1895 mL (IQR 1300-2500) in patients undergoing modified subtotal hysterectomy, and 260 min (IQR 210-287) and 2900 mL (IQR 2150-5500) in patients treated with total hysterectomy, respectively. The complication rate was 20% for MSHT and 82.3% for patients with a total hysterectomy. CONCLUSIONS: PAS in the cervical trigonal area associated with fibrosis implies a greater risk of complications due to uncontrollable bleeding and organ damage. MSTH is associated with lower morbidity and difficulties in PAS type 4. Prenatal or intrasurgical diagnosis is essential to plan surgical alternatives to improve the results.

Onabotulinum Toxin A Intradetrusor Injections in Children with Neurogenic Lower Urinary Tract Dysfunction: Long-Term Histological Effects on the Bladder Wall.

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.

Expression profiles of circular RNAs and interaction networks of competing endogenous RNAs in neurogenic bladder of rats following suprasacral spinal cord injury.

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

YAP/Smad3 promotes pathological extracellular matrix microenviroment-induced bladder smooth muscle proliferation in bladder fibrosis progression.

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.