ß-Adrenoceptors regulate urothelial inflammation and zonula occludens in the bladder outlet obstruction model.

BACKGROUND: The purpose of this study was to evaluate the effects of ß-adrenoceptors (ADRBs) on the urothelial inflammation and zonula occludens (ZO) in a rat PBOO model and in an in vitro model. METHODS: The PBOO model was established by ligating the bladder neck of rats. Twenty rats were divided into 4 groups: sham operation, PBOO + normal saline, PBOO + ADRB2 agonist, PBOO + ADRB3 agonist. PBOO rats were with treated with ADRBs agonists for 3 weeks. Human urothelial cells (HUCs) were subjected to ADRBs agonist treatment or hydrostatic pressure in an in vitro model. RESULTS: In the PBOO group, there was a significant increase in the expression of MCP-1, IL-6 and RANTES compared to the sham group. By contrast, there was a post-PBOO decline in the expression of ZO-1 and ZO-2 in the urothelium. ADRB2 or ADRB3 agonists exhibited downregulated inflammatory cytokine expression and increased ZO expression in the PBOO model. The regulation of inflammation and ZO by ADRB2 and ADRB3 agonists in an in vitro model was found consistent with that in the PBOO model. Moreover, RhoA and ROCK inhibitors suppressed the expression of hydrostatic pressure-induced inflammatory cytokines. Additionally, RhoA agonist reversed the inhibitory effect of ADRBs agonists on the inflammatory secretion from HUCs. CONCLUSIONS: ADRB2 and ADRB3 agonists increased ZO protein expression in HUCs in a rat PBOO model and in an in vitro model. Furthermore, ADRB2 and ADRB3 agonists inhibited the secretion of inflammatory cytokines from HUCs by regulating the RhoA/ROCK signaling pathways.

Excitatory purinergic and cholinergic expression changed in a partial bladder outlet obstruction-induced overactive bladder rat model.

Overactive bladder (OAB) is a common, long-term symptom complex with a high prevalence in women worldwide. OAB has caused a social burden, and effective treatments are urgently needed. However, the pathogenesis of OAB has yet to be elucidated. Model rats underwent bladder outlet obstruction surgery. In the 2nd, 3rd, and 4th weeks after surgery, metabolic cages were used to detect the 12 h urine volume of rats in the sham and model groups. The urodynamic parameters bladder leak point pressure (BPLL), maximum voiding pressure (MVP), residual volume (RV), maximum bladder capacity (MBC), bladder compliance (BC), voided efficiency (VE), and non-voiding contractions (NVCs) were also detected. Moreover, the contractile responses of isolated detrusor muscles to electrical and carbachol stimulation were examined at the abovementioned time points. At the 4th week after surgery, the bladders of both groups were obtained for hematoxylin-eosin (H&E) and Masson's trichrome staining. Real-time qPCR and Western blot were performed to quantify the expression of choline acetyltransferase (ChAT) and solute carrier family 17 member 9 (SLC17A9). At week 4, compared with the sham group, the 12 h urine volume of PBOO group increased significantly. The BLPP, MVP, VE, MBC, and NVCs increased significantly, and the VE was significantly reduced in 4-week PBOO group. The contractile responses of isolated detrusor muscles to electrical and carbachol stimulation significantly increased in 4-week PBOO group. In the 4-week PBOO group, the bladder wall and the ratio of bladder muscle to collagen within the bladder smooth muscle layer wall were significantly higher than those in the sham group. ChAT and SLC17A9 mRNA and protein expression in the OAB model rats significantly increased. At 4 weeks after PBOO, the OAB model was successfully established. The gene and protein expression levels of ChAT and SLC17A9 increased in the bladder of the OAB model, suggesting that OAB may be related to increased excitatory purinergic and cholinergic expression.

Molecular Characterization of Non-Neurogenic and Neurogenic Lower Urinary Tract Dysfunction (LUTD) in SCI-Induced and Partial Bladder Outlet Obstruction Mouse Models.

We examined bladder function following spinal cord injury (SCI) by repeated urodynamic investigation (UDI), including external urethral sphincter (EUS) electromyography (EMG) in awake restrained mice and correlated micturition parameters to gene expression and morphological changes in the bladder. A partial bladder outlet obstruction (pBOO) model was used for comparison to elucidate both the common and specific features of obstructive and neurogenic lower urinary tract dysfunction (LUTD). Thirty female C57Bl/6J mice in each group received an implanted bladder catheter with additional electrodes placed next to the EUS in the SCI group. UDI assessments were performed weekly for 7 weeks (pBOO group) or 8 weeks (SCI group), after which bladders were harvested for histological and transcriptome analysis. SCI mice developed detrusor sphincter dyssynergia (DSD) one week after injury with high-pressure oscillations and a significantly increased maximal bladder pressure Pmax and were unable to void spontaneously during the whole observation period. They showed an increased bladder-to-bodyweight ratio, bladder fibrosis, and transcriptome changes indicative of extracellular matrix remodeling and alterations of neuronal signaling and muscle contraction. In contrast, pBOO led to a significantly increased Pmax after one week, which normalized at later time points. Increased bladder-to-bodyweight ratio and pronounced gene expression changes involving immune and inflammatory pathways were observed 7 weeks after pBOO. Comparative transcriptome analysis of SCI and pBOO bladders revealed the activation of Wnt and TGF-beta signaling in both the neurogenic and obstructive LUTD and highlighted FGF2 as a major upregulated transcription factor during organ remodeling. We conclude that SCI-induced DSD in mice leads to profound changes in neuronal signaling and muscle contractility, leading to bladder fibrosis. In a similar time frame, significant bladder remodeling following pBOO allowed for functional compensation, preserving normal micturition parameters.

Upregulation of FGF7 Induced Intravesical Prostatic Protrusion of Benign Prostatic Hyperplasia via the ERK1/2 Signaling Pathway.

INTRODUCTION: Intravesical prostatic protrusion (IPP) has been reported to be associated with bladder outlet obstruction and is the main cause of lower urinary tract symptoms (LUTS) during the development of benign prostatic hyperplasia (BPH). However, the molecular mechanism of IPP remains unclear. METHODS: Clinical data analysis was performed to analyze the association between IPP and long-term complications in patients with BPH. RNA sequencing was performed on prostate tissues (IPP or not). Stromal cells were obtained from IPP-derived primary cultures to explore the molecular mechanism of IPP formation. Cell proliferation was evaluated by a CCK-8 assay. Multiple proteins in the signaling pathway were assessed using Western blot. RESULTS: First, we confirmed that IPP is a prognostic factor for long-term complications in patients with BPH. Then, we observed that FGF7 was upregulated in both IPP tissues and IPP primary stromal cells through immunohistochemistry, Western blot, and quantitative real-time PCR. Furthermore, FGF7 was significantly upregulated in high IPP-grade prostate tissues. The coculture experiments showed that the downregulation of FGF7 in IPP-derived stromal cells inhibited the proliferation and migration of the prostate epithelial cells. Additionally, FGF7 was bound to FGFR2 to induce the epithelial-mesenchymal transition process through binding to FGFR2. RNA sequencing analysis also revealed the activation of the MAPK/ERK1/2 signaling pathway. The MAPK/ERK1/2 was downregulated by a specific inhibitor affecting the FGF7 stimulation in vitro. CONCLUSIONS: Our data reveal a novel amplification effect, i.e., stromal cell-derived FGF7 promotes epithelial cell proliferation and stromal cell phenotype, ultimately inducing IPP formation. Targeting FGF7 can significantly reduce epithelial to stromal transition and provide a potential therapeutic target for BPH progression.

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.

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.

Molecular and Morphological Characteristics of the De-Obstructed Rat Urinary Bladder-An Update.

Many patients with outlet obstruction secondary to prostatic enlargement have lower urinary tract symptoms (LUTSs) and an increased frequency of micturition. The standard treatment is transurethral resection of the prostate (TURP), which alleviates obstruction and symptoms. However, after TURP, 20-40 percent of patients continue to experience LUTSs. The aim of the present study in rats was to identify the mechanisms that do not normalize after the removal of the obstruction and that could explain the persisting symptoms. We had microarray data from control, obstructed, and de-obstructed female rat bladders, which made it possible to study 14,553 mRNA expressions. We also had a bank of electron micrographs from similar detrusors. Microarrays: There were significant differences between the control and obstructed bladders for 1111 mRNAs. The obstructed and de-obstructed bladders differed significantly for 1059 mRNAs. The controls and the de-obstructed bladders differed significantly for 798 mRNAs. We observed many mRNAs that were increased in the obstructed bladder and then decreased to control levels after de-obstruction, and many mRNAs that were decreased in the obstructed bladder and then increased following de-obstruction. mRNAs that were significantly higher or lower in the de-obstructed bladder than in the control bladder were also found. Ultrastructure: The detrusor cells in the obstructed bladders had cross-sectional areas that were much larger than those in the controls. The control cells had smooth outlines and similar cross-sectional areas. The de-obstructed detrusor cells had larger cross-sectional areas than the controls, as well as corrugated surfaces. The cell areas varied, suggesting that the shrinkage of the de-obstructed cells was not even. We did not find any points of contact of the gap junction plaque type between the detrusor cells. There were abundant finger-like processes between the detrusor cells in the obstructed and in de-obstructed bladders, which were only occasionally found in the control detrusors. They are the only possible localization for gap junction channels. The de-obstructed rat bladder is not an organ with properties intermediate between those of the control and obstructed bladders. Instead, de-obstructed bladders have gene expressions, morphologies, and functional properties of the individual cells and their organization, which make them distinctly different from both control and obstructed bladders.

Astragaloside IV Protects Detrusor from Partial Bladder Outlet Obstruction-Induced Oxidative Stress by Activating Mitophagy through AMPK-ULK1 Pathway.

Aims: Bladder outlet obstruction (BOO) and the consequent low contractility of detrusor are the leading causes of voiding dysfunction. In this study, we aimed to evaluate the pharmacological activity of astragaloside IV (AS-IV), an antioxidant biomolecule that possess beneficial effect in many organs, on detrusor contractility and bladder wall remodeling process. Methods: Partial BOO (pBOO) was created by urethral occlusion in female rats, followed by oral gavage of different dose of AS-IV or vehicle. Cystometric evaluation and contractility test were performed. Bladder wall sections were used in morphology staining, and bladder tissue lysate was used for ELISA assay. Primary smooth muscle cells (SMCs) derived from detrusor were used for mechanism studies. Results: Seven weeks after pBOO, the bladder compensatory enlarged, and the contractility in response to electrical or chemical stimuli was reduced, while AS-IV treatment reversed this effect dose-dependently. AS-IV also showed beneficial effect on reversing the bladder wall remodeling process, as well as reducing ROS level. In mechanism study, AS-IV activated mitophagy and alleviated oxidative stress via an AMPK-dependent pathway. Conclusion: Out data suggested that AS-IV enhanced the contractility of detrusor and protected the bladder from obstruction induced damage, via enhancing the mitophagy and restoring mitochondria function trough an AMPK-dependent way.

Role of human adipose-derived stem cells (hADSC) on TGF-ß1, type I collagen, and fibrosis degree in bladder obstruction model of Wistar rats.

INTRODUCTION: Bladder outlet obstruction (BOO) was caused by a series of histological and biochemical changes in the bladder wall, through the inflammation process in the bladder wall, hypertrophy and fibrosis. ADSC has an important role in bladder regeneration. METHODS AND MATERIALS: This study was an experimental randomized study using male Wistar rats which were monitored at 2 and 4 weeks to determine the effect of ADSC therapy on TGF-ß1 type I collagen, and degree of fibrosis. RESULT: Rats were divided into 5 groups. In the week 2 BOO group, 1 sample included in the category of moderate fibrosis, 1 sample that was given ADSC with mild fibrosis category, 3 samples included in severe fibrosis category, 3 samples that were given ADSC included in the category of moderate fibrosis. The concentration of TGF-ß1 in the hADSC therapy group was significantly lower than the control group at the 2nd and 4th week of monitoring (p2 = 0.048, p4 = 0.048), and also with more type I collagen on 2nd and the 4th week (p2 = 0.048, p4 = 0.048). CONCLUSION: ADSC therapy can reduce the concentration of TGF-ß1, type I collagen, and degree of fibrosis in the male Wistar BOO model.

Enhanced Hypoxia-Associated Genes in Impaired Contractility From Bladder Outlet Obstruction.

BACKGROUND: Hypoxia damages the bladder wall and contributes to the initiation of bladder dysfunction. The change of hypoxia is not well known in impaired bladder contractility caused by long-term bladder outlet obstruction (BOO). We aimed to find out whether hypoxia of bladder tissue is present and what signaling mechanisms are involved in the decompensated bladder in BOO. METHODS: Twenty 6-week-old female Sprague-Dawley rats were divided into 2 groups, 10 rats each: group 1, sham operation; group 2, BOO for 8 weeks. Eight weeks after the onset of BOO, we did cystometric evaluation and processed polymerase chain reaction (PCR) array for hypoxia pathway using bladder tissues. The PCR array consists of 84 genes known to be involved in the hypoxic response, cell differentiation, and metabolism. We did quantitative PCR (qPCR) and immunohistochemical staining of bladder tissue for hypoxia. RESULTS: Eight genes were at least 2-fold upregulated and 3 genes were at least 2-fold downregulated in BOO group, compared with the sham operation group. The up-regulated genes (fold change) belonging to the hypoxia-inducible factor (HIF) 1 interactor included Cdkn2a (11.0), and the down-regulated genes belonging to HIF and co-transcription factors included Hif3a (-39.6) and Per1 (-5.1) by BOO. Genes influenced each other by means of TGFß1, TNF, and TP53. CONCLUSION: Hypoxia genes were increased in impaired contractility because of long-term BOO. The gene expression profiles could explain the molecular mechanisms of hypoxia in impaired contractility because of long-term BOO.

Human Urine-Derived Stem Cells Improve Partial Bladder Outlet Obstruction in Rats: Preliminary Data and microRNA-mRNA Expression Profile.

Partial bladder outlet obstruction (pBOO) often results in bladder tissue inflammation and remodeling. As human urine-derived stem cells (USCs) have demonstrated therapeutic benefits, we used a rat model to investigate the effect of USCs on bladder function and explore the miRNA and gene expression profiles in bladder tissue using RNA sequencing. Eighteen rats were assigned to a sham surgery group, pBOO group, and pBOO+USC group (six biweekly treatments). Routine urodynamic monitoring, analysis of detrusor muscle strips, and pathophysiology assessments were conducted. Finally, altered miRNA and mRNA expression profiles of bladder tissue were examined using RNA sequencing and bioinformatics analysis. After USC treatment, elevated bladder compliance and maximal voiding pressure, declined end filling pressure and voided volume, and improved detrusor muscle contractility and carbachol sensitivity were found. Histology and TUNEL assay revealed reduced collagen deposition and muscle cell apoptosis in bladder tissue. The differential expression of eight miRNAs was reversed by USC treatment. Two large nodes (miR-142 and miR-9a) were identified in the miRNA-gene interaction network in the USC-treated group. The Kyoto Encyclopedia of Genes and Genomes analysis revealed enrichment of multiple significant pathways, including those involved in necroptosis and cytokine-cytokine receptor interactions. This is the first study to demonstrate the protective effect of USCs on bladder function and remodeling in pBOO rats. The miRNA and mRNA expression levels differed in the bladder of pBOO rats with and without USC treatment. Although the mechanism underlying these effects has not been fully elucidated, necroptosis and cytokine-cytokine receptor interaction-related pathways may be involved.

Therapeutic effect of adipose stromal vascular fraction spheroids for partial bladder outlet obstruction induced underactive bladder.

BACKGROUND: Underactive bladder (UAB) is a common clinical problem but related research is rarely explored. As there are currently no effective therapies, the administration of adipose stromal vascular fraction (ad-SVF) provides a new potential method to treat underactive bladder. METHODS: Male Sprague-Dawley rats were induced by partial bladder outlet obstruction (PBOO) for four weeks and randomly divided into three groups: rats treated with PBS (Sham group); rats administrated with ad-SVF (ad-SVF group) and rats performed with ad-SVF spheroids (ad-SVFsp group). After four weeks, urodynamic studies were performed to evaluate bladder functions and all rats were sacrificed for further studies. RESULTS: We observed that the bladder functions and symptoms of UAB were significantly improved in the ad-SVFsp group than that in the Sham group and ad-SVF group. Meanwhile, our data showed that ad-SVF spheroids could remarkably promote angiogenesis, suppress cell apoptosis and stimulate cell proliferation in bladder tissue than that in the other two groups. Moreover, ad-SVF spheroids increased the expression levels of bFGF, HGF and VEGF-A than ad-SVF. IVIS Spectrum small-animal in vivo imaging system revealed that ad-SVF spheroids could increase the retention rate of transplanted cells in bladder tissue. CONCLUSIONS: Ad-SVF spheroids improved functions and symptoms of bladder induced by PBOO, which contributes to promote angiogenesis, suppress cell apoptosis and stimulate cell proliferation. Ad-SVF spheroids provide a potential treatment for the future patients with UAB.

Serum/glucocorticoid-regulated kinase 1-targeted transient receptor potential oxalate subtype 1 regulates bladder smooth muscle cell proliferation due to bladder outlet obstruction in mice via activated T cell nuclear factor transcription factor 2.

Bladder outlet obstruction (BOO) is a type of chronic disease that is mainly caused by benign prostatic hyperplasia. Previous studies discovered the involvements of both serum/glucocorticoid-regulated kinase 1 (SGK1) and activated T cell nuclear factor transcription factor 2 (NFAT2) in the proliferation of smooth muscle cells after BOO. However, the relationship between these two molecules is yet to be explored. Thus, this study explored the specific mechanism of the SGK1-NFAT2 signaling pathway in mouse BOO-mediated bladder smooth muscle cell proliferation in vivo and in vitro. In vivo experiments were performed by suturing 1/2 of the external urethra of female BALB/C mice to cause BOO for 2 weeks. In vitro, mouse bladder smooth muscle cells (MBSMCs) were treated with dexamethasone (Dex) or dexamethasone + SB705498 for 12 h and were transfected with SGK1 siRNA for 48 h. The expression and distribution of SGK1, transient receptor potential oxalate subtype 1 (TRPV1), NFAT2, and proliferating cell nuclear antigen (PCNA) were measured by Western blotting, polymerase chain reaction, and immunohistochemistry. The relationship between SGK1 and TRPV1 was analyzed by coimmunoprecipitation. The proliferation of MBSMCs was examined by 5-ethynyl-2'-deoxyuridine and cell counting kit 8 assays. Bladder weight, smooth muscle thickness, and collagen deposition in mice after 2 weeks of BOO were examined. Bladder weight, smooth muscle thickness, the collagen deposition ratio, and the expression of SGK1, TRPV1, NFAT2, and PCNA were significantly increased in mice after 2 weeks of BOO. Compared with the control, 10 µM Dex promoted the expression of these four molecules and the proliferation of MBSMCs. After inhibiting TRPV1, only the expression of SGK1 was not affected, and the proliferation of MBSMCs was inhibited. After silencing SGK1, the expression of these four molecules and the proliferation of MBSMCs decreased. Coimmunoprecipitation suggested that SGK1 acted directly on TRPV1. In this study, SGK1 targeted TRPV1 to regulate the proliferation of MBSMCs mediated by BOO in mice through NFAT2 and then affected the process of bladder remodeling after BOO. This finding may provide a strategy for BOO drug target screening.

Increased transient receptor potential canonical 3 activity is involved in the pathogenesis of detrusor overactivity by dynamic interaction with Na+/Ca2+ exchanger 1.

Transient receptor potential canonical 3 (TRPC3) is a nonselective cation channel, and its dysfunction is the basis of many clinical diseases. However, little is known about its possible role in the bladder. The purpose of this study was to explore the function and mechanism of TRPC3 in partial bladder outlet obstruction (PBOO)-induced detrusor overactivity (DO). We studied 31 adult female rats with DO induced by PBOO (the DO group) and 40 sham-operated rats (the control group). Here we report that the expression of TRPC3 in the bladder of DO rats increased significantly. Furthermore, PYR10, which can selectively inhibit the TRPC3 channel, significantly reduced bladder excitability in DO and control rats, but the decrease of the bladder excitability of DO rats was more obvious. PYR10 significantly reduced the intracellular calcium concentration in smooth muscle cells (SMCs) in DO and control rats. Finally, Na+/Ca2+ exchanger 1 (NCX1) colocalizes with TRPC3 and affects its expression and function. Collectively, these results indicate that TRPC3 plays an important role in the pathogenesis of DO through a synergistic effect with NCX1. TRPC3 and NCX1 may be new therapeutic targets for DO.

Specialized proresolution mediators in the bladder: annexin-A1 normalizes inflammation and bladder dysfunction during bladder outlet obstruction.

Bladder outlet obstruction (BOO) is ultimately experienced by ≈90% of men, most commonly secondary to benign prostatic hyperplasia. Inflammation is a critical driver of BOO pathology in the bladder and can be divided into two critical steps: initiation and resolution. Although great strides have been made toward understanding the initiation of inflammation in the bladder [through the NLR family pyrin domain containing 3 (NLRP3) inflammasome], no studies have examined resolution. Resolution is controlled by five classes of compounds known as specialized proresolving mediators (SPMs), all of which bind to one or more of the seven different receptors. Using immunocytochemistry, we showed the presence of six of the known SPM receptors in the bladder of control and BOO rats; the seventh SPM receptor has no rodent homolog. Expression was predominantly localized to urothelia, often with some expression in smooth muscle, but little to none in interstitial cells. We next examined the therapeutic potential of the annexin-A1 resolution system, also present in control and BOO bladders. Using the peptide mimetic Ac2-26, we blocked inflammation-initiating pathways (NLRP3 activation), diminished BOO-induced inflammation (Evans blue dye extravasation), and normalized bladder dysfunction (urodynamics). Excitingly, Ac2-26 also promoted faster and more complete functional recovery after surgical deobstruction. Together, the results demonstrate that the bladder expresses a wide variety of potential proresolving pathways and that modulation of just one of these pathways can alleviate many detrimental aspects of BOO and speed recovery after deobstruction. This work establishes a precedent for future studies evaluating SPM effectiveness in resolving the many conditions associated with bladder inflammation.NEW & NOTEWORTHY To our knowledge, this is the first study of proinflammation-resolving pathways in the bladder, which is the basis of a new pharmacological genus-dubbed "resolution pharmacology" aimed at reducing inflammation without creating an immunocompromised state. Inflammation plays a causative or exacerbating role in numerous bladder maladies. We documented proresolution receptors in the rat bladder and the effectiveness of a specialized proresolving mediator, annexin-A1, in alleviating detrimental aspects of bladder outlet obstruction and speeding recovery after deobstruction.

The roles of MCP-1/CCR2 mediated macrophage recruitment and polarization in bladder outlet obstruction (BOO) induced bladder remodeling.

Bladder outlet obstruction (BOO) can lead to alternation of bladder structure and function, known as bladder remodeling. Macrophage is a heterogeneous cell type and implicated in immunity regulating and tissue repairment. The relationship between macrophage and BOO remains unclear. We determined the pivotal role of macrophage recruitment and polarization in bladder remodeling. Sprague-Dawley rats underwent surgical operation of a BOO for either 1, 3, 6 weeks and were compared with sham-operated rats. The BOO rats in the experimental group were orally administrated with 5 mg/kg RS-504393, a C-C chemokine receptor (CCR2) antagonist, for 6 weeks, and the rats in the control group were treated with vehicle. Bladder tissues were harvested for assays of flow cytometry, quantitative reverse transcription polymerase chain reaction, histological examinations, immunohistochemistry staining and immunofluorescence. After induction of BOO, M1 macrophages were predominantly observed at inflammatory stage while M2 macrophages were mainly found during fibrosis stage. Flow cytometry analysis revealed that the ratio of M1/M2 significantly increased at 3 weeks (P = 0.0013) when compared to the sham-operated group. Interestingly, our results showed that M2 macrophages promoted BOO-induced fibrosis through indirectly secreting TGF-ß and directly transforming to collagen-producing myofibroblast. Additionally, RS-504393 treatment significantly decreased the number of M1 and M2 macrophage infiltration in bladder tissue, and bladder fibrosis was attenuated by RS-504393 treatment compared with that in the vehicle-treated rats. In summary, macrophages play a pivotal role in bladder remodeling and targeting MCP-1/CCR2 signaling pathway might be a therapeutic strategy for human 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.

Gene expression profiles during tissue remodeling following bladder outlet obstruction.

Bladder outlet obstruction (BOO) often results in lower urinary tract symptoms (LUTSs) and negatively affects quality of life. Here, we evaluated gene expression patterns in the urinary bladder during tissue remodeling due to BOO. We divided BOO model rats into two groups according to the degree of hypertrophy of smooth muscle in the bladder. The strong muscular hypertrophy group, which exhibited markedly increased bladder smooth muscle proportion and HIF1α mRNA levels compared with the control group, was considered a model for the termination of hypertrophy, whereas the mild muscular hypertrophy group was considered a model of the initiation of hypertrophy. Some genes related to urinary function showed different expression patterns between the two groups. Furthermore, we found that several genes, including D-box binding PAR bZIP transcription factor (DBP), were upregulated only in the mild muscular hypertrophy group. DBP expression levels were increased in bladder smooth muscle cells in response to hypoxic stress. DBP associated with enhancer and promoter regions of NOS3 gene locus and upregulated NOS3 gene expression under hypoxic conditions. These findings suggested that the regulatory systems of gene expression were altered during tissue remodeling following BOO. Furthermore, circadian clock components might be involved in control of urinary function via transcriptional gene regulation in response to hypoxic stimuli.

Metformin ameliorates bladder dysfunction in a rat model of partial bladder outlet obstruction.

Alteration of bladder morphology and function was the most important consequence of bladder outlet obstruction (BOO). Using a rat model of partial BOO (pBOO), we found that rats treated with metformin showed lower baseline pressures with a reduced inflammatory reaction in the early phase (2 wk) after pBOO. The NLR family pyrin domain containing 3 inflammasome pathway was inhibited in pBOO rat bladders with treatment of metformin in the early phase. Metformin reduced the activity of NLR family pyrin domain containing 3 in primary urothelial cells. In the chronic phase (9 wk after pBOO), metformin treatment ameliorated bladder fibrosis and improved the reduced compliance. Treatment with metformin suppressed the activation of Smad3 and compensated the diminished autophagy in 9-wk pBOO rat bladders. Autophagy was inhibited with upregulation of profibrotic proteins in primary fibroblasts from chronic pBOO bladders, which could be restored by administration of metformin. The antifibrotic effects of metformin on fibroblasts were diminished after silencing of AMP-activated protein kinase or light chain 3B. In summary, this study elucidates that oral administration of metformin relieves inflammation in the bladder during the early phase of pBOO. Long-term oral administration of metformin can prevent functional and histological changes in the pBOO rat bladder. The current study suggests that metformin might be used to prevent the development of bladder dysfunction secondary to BOO.NEW & NOTEWORTHY The present study in a rat model showed that oral administration of metformin alleviated inflammation following partial bladder outlet obstruction in the early phase and ameliorated bladder fibrosis as well as bladder dysfunction by long-term treatment. Our study indicated that metformin is a potential drug to inhibit bladder remodeling and alleviate bladder dysfunction. Clinical trials are needed to validate the effect of metformin on the bladder dysfunction and bladder fibrosis in the future.

The role of interleukin-6/interleukin-6 receptor signaling in the mechanical stress-induced extracellular matrix remodeling of bladder smooth muscle.

Extracellular matrix (ECM) remodeling is strongly associated with pathological changes induced by bladder outlet obstruction (BOO). In this study, we investigated the role of interleukin-6 (IL-6) in mechanical stretch-induced ECM remodeling of bladder smooth muscle. To construct a BOO animal model, the urethras of female Sprague-Dawley rats were partially ligated. In addition, increased hydrostatic pressure and mechanical stretching were applied to human bladder smooth muscle cells (HBSMCs) as an in vitro model. The expression of rat inflammatory genes was analyzed using DNA microarrays. We used quantitative RT-PCR (qRT-PCR) and immunohistochemical staining to detect IL-6 in the bladder smooth muscle of rats. To determine the specificity of IL-6, small interfering ribonucleic acid (siRNA) transfection and IL-6 receptor inhibitor (SC144) were applied to HBSMCs. qRT-PCR with siRNA transfection was also used to determine the specificity of downstream signaling. Moreover, western blotting was conducted to verify the expression results. In the animal model, the expression of ECM components and inflammatory genes was significantly upregulated. The expression of IL-6 was increased at both the mRNA level and the protein level in BOO rats. In vitro, hydrostatic pressure, and mechanical stretching both promoted MMP7 and MMP11 expression. Additionally, downregulation of collagen III occurred in both the hydrostatic pressure group and the mechanical stretch group. However, the expression of fibronectin exhibited opposing patterns between the hydrostatic pressure and mechanical stretch groups. The application of targeted siRNA transfection and an inhibitor (SC144) that targeted IL-6 significantly reversed the changes in MMP7 and MMP11 under mechanical stress and partially increased the expression of collagen III and fibronectin. In summary, IL-6 participated in the ECM remodeling of HBSMCs under mechanical stress, indicating that IL-6 may play an essential role in BOO..