Search

Long-term administration of exogenous estrogen is known to cause urinary retention and marked, often fatal, bladder distention in both male and female mice. Estrogen-treated mice have increased bladder pressure and decreased urine flow, suggesting that urinary retention in estrogen-treated mice is due to infravesicular obstruction to urine outflow. Thus, the condition is commonly referred to as bladder outlet obstruction (BOO). Obesity can also lead to urinary retention. As the effects of estrogen are mediated by multiple receptors, including estrogen receptors ERα and ERβ and the G protein-coupled estrogen receptor (GPER), we sought to determine whether GPER plays a role in estrogen-induced BOO, particularly in the context of obesity. Wild type and GPER knockout (KO) mice fed a high-fat diet were ovariectomized or left ovary-intact (sham surgery) and supplemented with slow-release estrogen or vehicle-only pellets. Supplementing both GPER KO and wild type obese mice with estrogen for 8 weeks resulted in weight loss, splenic enlargement, and thymic atrophy, as expected. However, estrogen-treated obese GPER KO mice developed abdominal distension, debilitation, and ulceration of the skin surrounding the urogenital opening. At necropsy, these mice had prominently distended bladders and hydronephrosis. In contrast, estrogen-treated obese wild type mice only rarely displayed these signs. Our results suggest that, under conditions of obesity, estrogen induces BOO as a result of ERα-driven pathways and that GPER expression is protective against BOO., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ERP and GS are inventors on U.S. patents, including those related to GPER-selective compounds (7875,721 and 8487,100) and their applications (10,251,870; 10,471,047; 10,561,648; 10,682,341; 10,980,785 and 11,963,949)., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Partial bladder outlet obstruction (pBOO) causes deposition of extracellular matrix (ECM), promotes bladder fibrosis, and decreases bladder compliance., Methods: To investigate the effect of β-adrenoceptor (ADRB) on the ECM deposition of pBOO rat model and explore its underlying mechanism, human bladder smooth muscle cells (hBSMCs) were exposed to the pathological hydrostatic pressure (100 cm H 2 O) for 6 h, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were employed. Then the rats of sham operation and pBOO model were treated with vehicle or ADRB agonists for 3 weeks, and the alterations of the bladder were observed via Masson staining and immunohistochemical analysis., Results: 100 cm H 2 O hydrostatic pressure significantly upregulated the expression of collagen I (COL1), collagen III (COL3) and fibronectin (FN), and downregulated the expression of ADRB2 and ADRB3 of hBSMCs at 6 h. The agonists of ADRB2 and ADRB3, Formoterol and BRL 37344, decreased COL1 and FN expression of hBSMCs under 100 cm H 2 O for 6 h compared with the cells exposed to hydrostatic pressure only. As the classic downstream pathways of ADRB, the EPAC pathway inhibited COL1 and FN expression of hBSMCs via regulating SMAD3 and SMAD2 activities, respectively. In pBOO rats, Procaterol (ADRB2 agonist), and Mirabegron (ADRB3 agonist) inhibited the formation of collagen and decreased the expression of FN and COL1 in the bladders of pBOO rats., Conclusions: The bladder fibrosis of pBOO and deposition of hBSMCs ECM under hydrostatic pressure were regulated by ADRB2, and ADRB3 via EPAC/SMAD2/FN and EPAC/SMAD3/COL1 pathways, these findings pave an avenue for effective treatment of pBOO., (© 2024 The Author(s). Published by IMR Press.)

Objective: Bladder outlet obstruction (BOO) results in significant fibrosis in the chronic stage and elevated bladder pressure. Piezo1 is a type of mechanosensitive (MS) channel that directly responds to mechanical stimuli. To identify new targets for intervention in the treatment of BOO-induced fibrosis, this study investigated the impact of high hydrostatic pressure (HHP) on Piezo1 activity and the progression of bladder fibrosis., Methods: Immunofluorescence staining was conducted to assess the protein abundance of Piezo1 in fibroblasts from obstructed rat bladders. Bladder fibroblasts were cultured under normal atmospheric conditions (0 cmH 2 O) or exposed to HHP (50 cmH 2 O or 100 cmH 2 O). Agonists or inhibitors of Piezo1, YAP1, and ROCK1 were used to determine the underlying mechanism., Results: The Piezo1 protein levels in fibroblasts from the obstructed bladder exhibited an elevation compared to the control group. HHP significantly promoted the expression of various pro-fibrotic factors and induced proliferation of fibroblasts. Additionally, the protein expression levels of Piezo1, YAP1, ROCK1 were elevated, and calcium influx was increased as the pressure increased. These effects were attenuated by the Piezo1 inhibitor Dooku1. The Piezo1 activator Yoda1 induced the expression of pro-fibrotic factors and the proliferation of fibroblasts, and elevated the protein levels of YAP1 and ROCK1 under normal atmospheric conditions in vitro. However, these effects could be partially inhibited by YAP1 or ROCK inhibitors., Conclusion: The study suggests that HHP may exacerbate bladder fibrosis through activating Piezo1., (© 2024. Huazhong University of Science and Technology.)

Bladder outlet obstruction (BOO) is the primary clinical manifestation of benign prostatic hyperplasia, the most common urinary system disease in elderly men, and leads to associated lower urinary tract symptoms. Although BOO is reportedly associated with increased systemic oxidative stress (OS), the underlying mechanism remains unclear. The elucidation of this mechanism is the primary aim of this study. A Sprague-Dawley rat model of BOO was constructed and used for urodynamic monitoring. The bladder tissue of rats was collected and subjected to real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), histological examination, and immunohistochemical staining. Through bioinformatics prediction, we found that transforming growth factor β2 (TGFβ2) expression was upregulated in rats with BOO compared with normal bladder tissue. In vitro analyses using primary bladder smooth muscle cells (BSMCs) revealed that hydrogen peroxide (H 2 O 2 ) induced TGFβ2 expression. Moreover, H 2 O 2 induced epithelial-to-mesenchymal transition (EMT) by reducing E-cadherin, an endothelial marker and CK-18, a cytokeratin maker, and increasing mesenchymal markers, including N-cadherin, vimentin, and α-smooth muscle actin (α-SMA) levels. The downregulation of TGFβ2 expression in BSMCs using siRNA technology alleviated H 2 O 2 -induced changes in EMT marker expression. The findings of the study indicate that TGFβ2 plays a crucial role in BOO by participating in OS-induced EMT in BSMCs., (© 2024. The Author(s).)

This study investigates whether hAFSCs can improve bladder function in partial bladder outlet obstruction (pBOO) rats by targeting specific cellular pathways. Thirty-six female rats were divided into sham and pBOO groups with and without hAFSCs single injection into the bladder wall. Cystometry, inflammation/hypoxia, collagen/fibrosis/gap junction proteins, and smooth muscle myosin/muscarinic receptors were examined at 2 and 6 weeks after pBOO or sham operation. In pBOO bladders, significant increases in peak voiding pressure and residual volume stimulated a significant upregulation of inflammatory and hypoxic factors, TGF-β1 and Smad2/3. Collagen deposition proteins, collagen 1 and 3, were significantly increased, but bladder fibrosis markers, caveolin 1 and 3, were significantly decreased. Gap junction intercellular communication protein, connexin 43, was significantly increased, but the number of caveolae was significantly decreased. Markers for the smooth muscle phenotype, myosin heavy chain 11 and guanylate-dependent protein kinase, as well as M2 muscarinic receptors, were significantly increased in cultured detrusor cells. However, hAFSCs treatment could significantly ameliorate bladder dysfunction by inactivating the TGFβ-Smad signaling pathway, reducing collagen deposition, disrupting gap junctional intercellular communication, and modifying the expressions of smooth muscle myosin and caveolae/caveolin proteins. The results support the potential value of hAFSCs-based treatment of bladder dysfunction in BOO patients.

Aim: This study aimed to investigate the clinical significance of neutrophil-to-lymphocyte ratio and ultrasonic parameters in diagnosing bladder outlet obstruction in patients with benign prostatic hyperplasia., Material: Between September 2022 and January 2024, a total of 106 patients with benign prostatic hyperplasia were collected from Hongqi Hospital affiliated to Mudanjiang Medical University followed by urodynamic testing. The patients were categorized into three groups based on the International Prostate Symptom Score: mild (45 cases), moderate (36 cases), and severe (25 cases). Thirty-five healthy men were recruited at the hospital as a control group. All patients had blood tests and ultrasound scans., Results: Neutrophil-to-lymphocyte ratio, detrusor wall thickness, detrusor muscle elastic modulus, internal gland elastic modulus, intravesical prostatic protrusion, and post-voiding residual volume were significantly correlated with the bladder outlet obstruction stage and showed good diagnostic efficiency (all P<0.05. There was no statistically significant difference observed in the external gland elastic modulus between the experimental group and the control group (P>0.05)., Conclusions: The neutrophil-to-lymphocyte ratio, detrusor wall thickness, elastic modulus of the detrusor and glandular gland may hold clinical significance for diagnosing bladder outlet obstruction resulting from benign prostatic hyperplasia., (Copyright © 2024. Published by Elsevier Masson SAS.)

Objectives: This study evaluates the impact of equol, a metabolite of soy isoflavone, on bladder dysfunction in rats with bladder outlet obstruction (BOO). In addition, we investigate its potential as a neuroprotective agent for the obstructed bladder and discuss its applicability in managing overactive bladder (OAB)., Methods: Eighteen male Sprague-Dawley rats were divided into three groups (six rats per group) during the rearing period. The Sham and C-BOO groups received an equol-free diet, while the E-BOO group received equol supplementation (0.25 g/kg). At 8 weeks old, rats underwent BOO surgery, followed by continuous cystometry after 4 weeks of rearing. The urinary oxidative stress markers (8-hydroxy-2'-deoxyguanosine and malondialdehyde) were measured, and the bladder histology was analyzed using hematoxylin-eosin, Masson's trichrome, and immunohistochemical staining (neurofilament heavy chain for myelinated nerves, peripherin for unmyelinated nerves, and malondialdehyde)., Results: Equol reduced BOO-induced smooth muscle layer fibrosis, significantly prolonged the micturition interval (C-BOO: 193 s, E-BOO: 438 s) and increased the micturition volume (C-BOO: 0.54 mL, E-BOO: 1.02 mL) compared to the C-BOO group. Equol inhibited the increase in urinary and bladder tissue malondialdehyde levels. While the C-BOO group exhibited reduced peripherin alone positive nerve fibers within the smooth muscle layer, equol effectively attenuated this decline., Conclusions: Equol reduces lipid peroxidation and smooth muscle layer fibrosis in the bladder and exhibited neuroprotective effects on bladder nerves (peripheral nerves) and prevented the development of bladder dysfunction associated with BOO in rats. Consumption of equol is promising for the prevention of OAB associated with BOO., (© 2024 John Wiley & Sons Australia, Ltd.)

Background: In older adults, bladder outlet obstruction (BOO) is prevalent, primarily due to benign prostatic hyperplasia (BPH). These patients' lower urinary tract symptoms can be treated surgically and with medical therapy. Compared to standard treatment with tamsulosin, Pentoxifylline, a phosphodiesterase inhibitor, could benefit patients with BOO due to its properties on microcirculatory blood flow and oxygenation of ischemic tissues. Hence, this trial intended to study the efficacy of Pentoxifylline combined with tamsulosin in treating BOO patients., Materials and Methods: This randomized, double-blind clinical trial recruited 60 patients with BPH from a single center in 2022. Upon consent of patients meeting the eligibility criteria, they were randomly allocated to intervention (Pentoxifylline + tamsulosin) and control (placebo + tamsulosin) groups. The patients were evaluated for international prostate symptom score (IPSS), quality of life (QoL), maximum urinary flow rate (Q max ) by uroflowmetry, and post-void residual volume (PVR) by abdominal sonography at the onset of the study and after the 12th week., Results: Patients who used the combination therapy had significantly better results of prostate symptoms and quality of life improvement (IPSS: -36.6%, QoL: -45.3%) compared to patients who received tamsulosin alone (IPSS: -21.2%, QoL: -27.7%) (p < .001). Also, this study shows that the improvement in maximum urinary flow rate and residual volume by combination therapy is significantly higher (Q max : +42.5%, PVR: -42.6%) compared to monotherapy (Q max : +25.1%, PVR: -26.1%) (p < .001)., Conclusion: When combined with tamsulosin, Pentoxifylline could significantly improve the lower urinary symptoms of BPH patients. It is well tolerated, and the treatment outcomes are better in patients who receive the combination of Pentoxifylline and tamsulosin than those who only receive tamsulosin., (© 2024 John Wiley & Sons Australia, Ltd.)

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 TGFβ1 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 TGFβ1-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 TGFβ1-induced BSMCs. Overexpression of NRF1 in hUSC-Exos further improved the effects of hUSC-Exos on bladder fibrosis both in vivo and in vitro. TGFβR1 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 TGFβR1level. Our results illustrated that hUSC-Exos carried NRF1 to alleviate bladder fibrosis through regulating miR-301b-3p/TGFβR1 pathway., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

We present a constrained mixture-micturition-growth (CMMG) model for the bladder. It simulates bladder mechanics, voiding function (micturition) and tissue adaptations in response to altered biomechanical conditions. The CMMG model is calibrated with both in vivo and in vitro data from healthy male rat urinary bladders (cystometry, bioimaging of wall structure, mechanical testing) and applied to simulate the growth and remodeling (G&R) response to partial bladder outlet obstruction (BOO). The bladder wall is represented as a multi-layered, anisotropic, nonlinear constrained mixture. A short time scale micturition component of the CMMG model accounts for the active and passive mechanics of voiding. Over a second, longer time scale, G&R algorithms for the evolution of both cellular and extracellular constituents act to maintain/restore bladder (homeostatic) functionality. The CMMG model is applied to a spherical membrane model of the BOO bladder utilizing temporal data from an experimental male rodent model to parameterize and then verify the model. Consistent with the experimental studies of BOO, the model predicts: an initial loss of voiding capacity followed by hypertrophy of SMC to restore voiding function; bladder enlargement; collagen remodeling to maintain its role as a protective sheath; and increased voiding duration with lower average flow rate. This CMMG model enables a mechanistic approach for investigating the bladder's structure-function relationship and its adaption in pathological conditions. While the approach is illustrated with a conceptual spherical bladder model, it provides the basis for application of the CMMG model to anatomical geometries. Such a mechanistic approach has promise as an in silico tool for the rational development of new surgical and pharmacological treatments for bladder diseases such as BOO., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The aim of this study is to investigate the incidental prostate cancer (iPCa) detection rates of different embedding methods in a large, contemporary cohort of patients with bladder outlet obstruction (BOO) treated with transurethral surgery. We relied on an institutional tertiary-care database to identify BOO patients who underwent either transurethral loop resection or laser (Holmium:yttrium-aluminium garnet) enucleation of the prostate (HoLEP) between 01/2012 and 12/2019. Embedding methods differed with regard to the extent of the additional prostate tissue submitted following the first ten cassettes of primary embedding (cohort A: one [additional] cassette/10 g residual tissue vs. cohort B: complete embedding of the residual tissue). Detection rates of iPCa among the different embedding methods were compared. Subsequently, subgroup analyses by embedding protocol were repeated in HoLEP-treated patients only. In the overall cohort, the iPCa detection rate was 11% (46/420). In cohort A (n = 299), tissue embedding resulted in a median of 8 cassettes/patient (range 1-38) vs. a median of 15 (range 2-74) in cohort B (n = 121) (p < .001). The iPCa detection rate was 8% (23/299) and 19% (23/121) in cohort A vs. cohort B, respectively (p < .001). Virtual reduction of the number of tissue cassettes to ten cassettes resulted in a iPCa detection rate of 96% in both cohorts, missing one stage T1a/ISUP grade 1 carcinoma. Increasing the number of cassettes by two and eight cassettes, respectively, resulted in a detection rate of 100% in both cohorts without revealing high-grade carcinomas. Subgroup analyses in HoLEP patients confirmed these findings, demonstrated by a 100 vs. 96% iPCa detection rate following examination of the first ten cassettes, missing one case of T1a/ISUP 1. Examination of 8 additional cassettes resulted in a 100% detection rate. The extent of embedding of material obtained from transurethral prostate resection correlates with the iPCa detection rate. However, the submission of 10 cassettes appears to be a reasonable threshold to reduce resource utilization while maintaining secure cancer detection., (© 2022. The Author(s).)

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-β1 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-β1 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-β1, type I collagen, and degree of fibrosis in the male Wistar BOO model., (© 2022. The Author(s).)

The effects of circular RNAs (circRNAs) on bladder outlet obstruction (BOO)-induced hypertrophy and fibrogenesis in rats and hypoxia-induced bladder smooth muscle cell (BSMC) fibrosis remain unclear. This study aimed to determine the regulatory role of circRNAs in the phenotypic changes in BSMCs in BOO-induced rats.circRNAmicroarray and real-time PCR were used to explore differentiated expressed circRNAs. Bioinformatics analyses and dual-luciferase reporter were performed to identify the targets for circRNA PVT1 (circPVT1). BOO was performed to establish a bladder fibrosis animal model. The circPVT1 and suppressor of cytokine signaling 3 (SOCS3) expression levels were upregulated (p = 0.0061 and 0.0328, respectively), whereas the microRNA-203a (miR-203) level was downregulated in rats with bladder remodeling (p=0.0085). Bioinformatics analyses and dual-luciferase reporter assay results confirmed that circPVT1 sponges miR-203 and that the latter targets the 3'-untranslated region of SOCS3. Additionally, circPVT1 knockdown alleviated BOO-induced bladder hypertrophy and fibrogenesis. Furthermore, hypoxia was induced in BSMCs to establish a cell model of bladder fibrosis. Hypoxia induction in BSMCs resulted in upregulated circPVT1 and SOCS3 levels (p = 0.0052) and downregulated miR-203 levels. Transfection with circPVT1 and SOCS3 shRNA ameliorated hypoxia-induced transforming growth factor-β (TGF-β1), TGFβR1, α-smooth muscle actin, fibrotic growth factor, extracellular matrix subtypes, BSMC proliferation, and apoptosis-associated cell injury, whereas co-transfection with miR-203 inhibitor counteracted the effect of circPVT1 shRNA on these phenotypes.These findings revealed a novel circRNA regulator of BOO-associated bladder wall remodeling and hypoxia-induced phenotypic changes in BMSCs by targeting the miR-203-SOCS3 signaling axis.

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.

Purpose: To determine the unique contributions from elevated voiding and storage pressures in the development of fibrosis and the epithelial-to-mesenchymal transition (EMT) in urothelial cells, and how progressive BOO pressure cycling is an important mechanical cue leading to these pathological changes., Materials and Methods: Urothelial cells isolated from control, SHAM, 2 (acute)- or 6 (chronic)-week BOO rats treated with an inflammasome inhibitor or no drug. Total RNA was isolated and RT-PCR was conducted with custom primers for pro-fibrotic and EMT genes. In separate experiments, a rat urothelial cell line was exposed to cyclic pressure regimes characteristic of acute and chronic BOO in the presence or absence of an inflammasome inhibitor. Following exposure, RT-PCR was conducted, collagen content was determined and intracellular caspase-1 activity was measured., Results: Urothelial cells isolated from acute and chronic BOO rat models demonstrated expression of pro-fibrotic and EMT genes. Similarly, MYP3 rat urothelial cells subjected to pressure cycling regimes that reflect intravesical pressures in the acute or chronic BOO bladder also demonstrated increased expression of pro-fibrotic and EMT genes, along with elevated soluble collagen. Treatment with inflammasome inhibitors reduced expression of pro-fibrotic genes in the rat model and pressure cycling model but had a limited effect on EMT., Conclusion: These results indicate that acute and chronic BOO pressure cycling are essential in the initiation and progression of fibrosis in the bladder via the NLRP3 inflammasome, but also provide new evidence that there is also an alternative NLRP3-independent pathway leading to EMT and fibrosis., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Purpose: George Marion first described primary bladder neck obstruction in 1933. Even today, the etiopathogenesis of this condition is unknown. The objective of this study is to associate a novel histologic finding with Marion's disease to contribute to its etiopathogenesis, and to seek the reason why the lower urinary tract symptoms of some patients was not relieved after pharmacological treatment., Methods: The retrospective study was carried out with patients who underwent transurethral resection of the prostate from 2009 to 2019. Patients with histological diagnosis did not present hyperplastic nodules, and the presence of skeletal muscle fibers were included in the study. The frequency of cases with presence of skeletal muscle fibers was assessed as well as the area occupied by these fibers in each resected fragment. As a control group, fragments of bladder neck of surgical specimens from 50 radical prostatectomies were analyzed., Results: In 14 patients with skeletal muscle fibers in the resected fragments the extent of each positive fragment was < 25%, > 25-50%, > 50-75%, and > 75% in 28.6%, 28.6%, 21.4%, and 21.4% fragments, respectively. In the control group, 20% (10/50) of the patients had skeletal muscle fibers and, in all cases, they occupied < 25%., Conclusion: In these 14 cases, the presence of skeletal muscle fibers is frequent and predominant in TURP fragments, which stands in striking contrast to the control group. We consider that presence of skeletal muscle fibers may be related to Marion's disease, thus contributing to explain its etiopathogenesis and the unsuccessful alpha-blocker treatment in these patients., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

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-β (TGF-β) 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-β 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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.
., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

Objective: To present the clinical and radiological characteristics of women with severe structural deterioration of the bladder and upper urinary tract secondary to Primary Bladder Neck Obstruction (PBNO), and their outcomes after bladder neck incision (BNI)., Methods: Retrospective evaluation of adult women who underwent BNI for PBNO at one institution. Patients were assessed for symptoms, renal function, structural abnormalities of the urinary tract and video-urodynamics. PBNO diagnosis was confirmed with video-urodynamics in all patients. BNI was performed at the 4-5 and/or 7-8 o'clock positions. Postoperative symptoms, PVR, uroflowmetry and renal function were evaluated and compared to baseline., Results: Median patient age was 56.5 years (range 40-80). All presented with urinary retention-four were on clean intermittent Catheterization (CIC) and two with a Foley catheter. All patients had bladder wall thickening and diverticula. Four women had elevated creatinine levels, bilateral hydronephrosis was present in five (83.3%). After BNI, all patients resumed spontaneous voiding without the need for CIC. Median Qmax significantly improved from 2.0 [1.0-4.0] mL/s to 15 [10-22.7] mL/s (p = 0.031). Median PVR decreased from 150 to 46 [22-76] mL (p = 0.031). There were no postoperative complications. Creatinine levels returned to normal in 3/4 (75%) patients., Conclusion: PBNO in women may result in severe damage to the bladder and upper urinary tract. Despite severe structural abnormalities of the bladder, BNI was effective in reducing symptoms and improving structural and functional abnormalities of the lower and upper urinary tract., Competing Interests: The authors have declared that no competing interests exist.

We aimed to investigate clinical features potentially useful in primary bladder neck obstruction (PBNO) diagnosis in men presenting with lower urinary tract symptoms (LUTS). Data from 1229 men presenting for LUTS as their primary complaint at a single centre were retrospectively analysed. All patients underwent a comprehensive medical and physical assessment, and completed the International Prostate Symptoms Score. All patients were investigated with uroflowmetry, and trans-rectal ultrasound imaging to define prostate volume. Urodynamic evaluation was performed when the diagnosis of benign prostatic enlargement was not confirmed and the patient presented a significant chance of detrusor overactivity or underactivity. As per our internal protocol, patients < 60 years old with bothersome LUTS and > 60 years with a prostate volume (PV) < 40 mL were also investigated with urethrocystoscopy to rule out urethral stricture. Logistic regression analysis tested clinical predictors of possible PBNO. Of 1229 patients, 136 (11%) featured a clinical profile which was consistent with PBNO. Overall, these patients were younger (p < 0.0001), had lower BMI (p < 0.0001), less comorbidities (p = 0.004) and lower PSA values (p < 0.0001), but worse IPSS scores (p = 0.01) and lower PV values (p < 0.0001) compared to patients with other-aetiology LUTS. At multivariable analysis, younger age (OR 0.90; p = 0.003) and higher IPSS scores (OR 1.12; p = 0.01) were more likely to be associated with this subset of patients, after accounting for other clinical variables. One out of ten young/middle-aged men presenting for LUTS may be affected from PBNO. Younger patients with more severe LUTS systematically deserve an extensive assessment to rule out PBNO, thus including urethrocystoscopy and urodynamics with voiding-cysto-urethrogram.

Purpose: We investigated the possible association between preoperative bladder wall thickness (BWT) or detrusor wall thickness (DWT) and bladder outlet obstruction (BOO) based on urodynamic studies in men with symptomatic benign prostatic hyperplasia (BPH)., Materials and Methods: Data were prospectively collected from a BPH surgery database. A total of 196 men who underwent prostate vaporization for symptomatic BPH were included in this study. BWT and DWT were measured in the suprapubic area after uroflowmetry., Results: No significant difference was noted in BWT and DWT in any patient according to the presence of BOO; however, subgroup analysis showed that BWT and DWT were significantly thicker in the obstruction group in men aged 70 years or older than in those under age 70 (BWT: 3.6+0.9 mm vs. 3.1+0.9 mm, p=0.022, DWT: 2.8±0.8 mm vs. 2.3±0.8 mm, p=0.007). In this older age group, the classification based on a BWT ≥4.0 mm showed 31% sensitivity, 87% specificity, and 65% diagnostic accuracy for the diagnosis of BOO, whereas DWT ≥3.0 mm showed 49% sensitivity, 82% specificity, and 69% diagnostic accuracy., Conclusions: BWT and DWT were associated with BOO in men aged 70 years or older. Therefore, BWT and DWT will be a useful non-invasive parameter for deciding the management strategy for elderly men with symptomatic BPH. An appropriate measurement method should be established as soon as possible for further application of the relationship among BWT, DWT and BOO., Competing Interests: The authors have nothing to disclose., (© The Korean Urological Association, 2020.)

Introduction: We investigated which benign prostatic hyperplasia-related lower urinary parameters are related to upper urinary tract obstruction and whether transurethral prostatectomy could improve upper urinary tract obstruction., Materials and Methods: Patients with prostate size over 30 g and urodynamically proven bladder outlet obstruction were enrolled in this prospective observational study. Bladder wall thickness and prostate size were measured by ultrasonography. A urodynamic study with laboratory tests including serum creatinine, prostate-specific antigen, and urinalysis was performed. Finally, a diuretic scintigraphy using mercaptoacetyltriglycine was performed. Tests except the urodynamic evaluation were repeated after transurethral prostatectomy., Results: In total, 24 patients were enrolled, and 19 patients completed the present study. The mean values of age (yrs), prostate size (mL), bladder thickness (mm), bladder compliance ( Δ mL/ Δ pr), and the bladder outlet obstruction index were 68.42 ± 8.25, 72.29 ± 32.78, 4.42 ± 1.14, 50.17 ± 32.15, and 82.11 ± 34.68, respectively. The mean T 1/2 (min) was 17.51 ± 16.34 on the left side and 15.30 ± 11.96 on the right side. Statistical analysis showed that bladder compliance and bladder thickness were preoperatively related to upper urinary tract obstruction ( p = 0.001 and p = 0.007, respectively). Diuretic mercaptoacetyltriglycine scan in 19 patients showed improvement 6 months after prostate surgery. Clinically significant proteinuria was associated with upper urinary tract obstruction, and proteinuria was also improved after prostate surgery., Conclusion: Storage-phase bladder dysfunction could be a reliable urodynamic factor for the indication of upper urinary tract obstruction in patients with benign prostatic hyperplasia, and upper urinary tract obstruction with subsequent kidney damage could be improved by surgical decompression of benign prostatic obstruction., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Sung Yong Cho et al.)

The circadian clock programs daily rhythms and coordinates multiple behavioural processes, including micturition. Partial bladder outlet obstruction (pBOO) in mice produces hyperactive voiding. However, long-term effects of pBOO on bladder function have not been clarified. In this study, we investigated micturition under conditions of impaired circadian bladder function by inducing long-term pBOO by tying the proximal urethra. Micturition behavior was evaluated at 1, 3, 6 and 12 months after surgery. We used automated voided stain on paper method for a precise micturition recording for mice. And quantitative assessment of gene expression was performed at 24 months after pBOO surgery using qRT-PCR procedure. The micturition frequencies in the pBOO group were significantly decreased at 3, 6, and 12 months compared to those at 1 month after operation in the same group (p < 0.05). Body weight of pBOO mice was significantly increased compared to sham operated mice at 12 months. The expression level of mRNA was exhibited a 3.4-fold nominal increased for a 5-HT2B receptor in the pBOO group compared to the sham group. The current study found that long-term pBOO led to disruption of the circadian bladder function (the day/night cycle) in mice, similar to those observed in human as nocturia. This disruption is possible involvement of the gain of body weight and/or serotonergic alteration after pBOO.

Congenital lower urinary tract obstruction is associated with oligohydramnios and significant perinatal mortality and long-term chronic kidney disease. The counseling of families facing this diagnosis, especially when prenatal intervention is proposed, is fraught with ambiguity. This review aims to equip the provider with the current evidence behind the conventional and novel biomarkers predictive of chronic kidney disease. The relevant clinical predictors are categorized by when they are identified, antenatally or postnatally, and as either anatomic or chemical. They are considered for their prognostic value and the challenges in obtaining them, specifically the risk to the fetus in the case of prenatal biomarkers. Serum creatinine in infancy is the traditional chemical biomarker of kidney function and continues to be a consistent predictor of future serum creatinine. β-2 microglobulin may provide earlier information regarding fetal glomerular and tubular function and is also predictive of long-term serum creatinine. Renal parenchymal area is an anatomic surrogate of nephron mass that is used in both prenatal and postnatal settings. Understanding the anatomic and chemical biomarkers is essential for future refinement of the staging algorithm used to distinguish which patients may benefit from early in utero intervention.

Bladder outlet obstruction (BOO), which is primarily caused by benign prostatic hyperplasia, is a common chronic disease. However, previous studies have most commonly investigated BOO using the acute obstruction model. In the present study, a chronic obstruction model was established to investigate the different pathological alterations in the bladder between acute and chronic obstruction. Compared with chronic obstruction, acute obstruction led to increased expression of proliferating cell nuclear antigen and interleukin‑1β, which are markers of proliferation and inflammation, respectively. Furthermore, increased fibrosis in the bladder at week 2 was observed. Low pressure promoted mice bladder smooth muscle cell (MBSMC) proliferation, and pressure overload inhibited cell proliferation and increased the proportion of dead MBSMCs. Further investigation using serum/glucocorticoid regulated kinase 1 (SGK1) small interfering RNAs indicated that low pressure may promote MBSMC proliferation by upregulating SGK1 and nuclear factor of activated T‑cell expression levels. Therefore, the present study suggested that acute obstruction led to faster decompensation of bladder function and chronic bladder obstruction displayed an enhanced ability to progress to BOO.

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., (© 2020 Wiley Periodicals LLC.)

A 66-year-old man was found dead at his home address. He had a history of poor nutrition with recent episodes of vomiting. At autopsy the decedent was cachexic with a body mass index (BMI) of 16.1. The major findings were of marked prostatomegaly (165 g) with obstruction to urine outflow resulting in dilatation and trabeculation of the bladder and bilateral hydroureteronephrosis. The bladder contained 1.5 l of yellow urine and the kidneys weighed only 109 g on the left and 125 g on the right (N = 140-160 g). Histological examination of the prostate revealed benign nodular hyperplasia. Biochemical analysis of vitreous humor demonstrated a creatinine of 1579 μmol/L (normal 45-90) and a urea of 133.4 mmol/L (normal 2.5-7.1). Despite genitourinary causes of sudden and/or unexpected death being rare in routine forensic practice, this case demonstrates significant underlying obstructive renal disease that resulted in sudden death and that had remained undiagnosed until the time of autopsy.

Purpose: Partial bladder outlet obstruction (PBOO) induces sustained bladder over-distension, leading to ischemia/reperfusion (I/R)-related oxidative damage of the urothelium via apoptosis. The present study aimed to investigate the sequential course of apoptosis in the urothelium of rat bladder and identify the changes in apoptosis-related proteins during PBOO and subsequent relief.  Materials and Methods: The study was conducted using 60 female Sprague-Dawley rats divided into three groups: sham-operated, PBOO only, and PBOO plus subsequent relief. PBOO was induced for 2 weeks, and then the obstruction was relieved by removal of the ligature. The urothelium was assessed by a histological analysis, and expression levels of apoptosis-related proteins were detected by quantitative PCR and immunoblotting., Results: Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells were significantly increased in the PBOO only group when compared with the sham-operated group, and decreased in the PBOO relief group when compared with the PBOO group (P < 0.001). From the quantitative PCR and the western blot analyses, expression of Bax, caspase-3, P38, and Jnk was significantly increased in the PBOO group (P < 0.001). However, expression of Erk, Bcl-2 significantly decreased in the PBOO group (P < 0.001). The expression of Erk and Bcl-2 significantly increased in the PBOO relief group when compared with the PBOO group (P < 0.001). In comparison to the sham-operated group, expression levels of survivin significantly increased in both the PBOO and PBOO plus relief groups (P < 0.001). In addition, the expression levels were significantly different between the PBOO and PBOO plus relief groups (P < 0.001)., Conclusion: PBOO induced apoptosis of urothelium is related to alterations in the MAPK signaling pathways and apoptosis-related protein change. These results may also suggest that the pro-survival Erk signaling cascade and the expression survivin are activated in response to ischemic bladder injury and associated with initiation of bladder restoration in PBOO and subsequent relief. However, the mechanism of survivin as anti-apoptotic protein in ischemic bladder injuries remains unclear.

Partial bladder outlet obstruction (pBOO) results in bladder fibrosis that is initiated by an inflammatory cascade and the decompensation after smooth muscle hypertrophy. We have been using an animal model to develop the hypothesis that mesenchymal stem cells (MSCs) are able to mitigate this cytokine cascade and prevent bladder deterioration. We hypothesized that intraperitoneal administration of MSCs can produce the same effects as intravenously administered cells but may require higher dosing. Intraperitoneal treatment will provide insights into the mechanisms of action and may offer advantages over intravenous administration, as it will permit allow higher doses and potentially reduce systemic exposure. Rats underwent a surgical induction of pBOO and instillation of either 1 × 10 6 or 5 × 10 6 commercially acquired MSCs into the peritoneum. RT-PCR, immunohistochemistry, and urodynamics were used to compare treatment groups with controls. pBOO resulted in a marked, statistically significant, upregulation of inflammatory markers in the bladder, including transforming growth factor-β, hypoxia-inducible factor-1α, hypoxia-inducible factor-3α, mammalian target of rapamycin, and collagen types I and III. Moderate but inconsistent levels of downregulation were seen with 1 × 10 6 MSCs, but excellent and reliable downregulation was seen with 5 × 10 6 MSCs ( P < 0.05). Immunohistochemistry confirmed that protein levels were affected in accordance with mRNA upregulation. Urodynamics demonstrated MSC treatment resulted in whole organ physiological benefits, as they prevented elevations in detrusor pressure. In conclusion, intraperitoneal administration of MSCs resulted in a similar effect as intravenous administration; however, this required a higher dose. This has significant implications for determining the mechanism of action and potential clinical application for human therapy.

Current and potential medical therapy for obstruction-induced myopathic bladder dysfunction (from benign prostatic hyperplasia or posterior urethral valves) focuses on symptoms. The persistent tissue pathology and dysfunction after release of obstruction is often deemed irreversible without any systematic therapeutic approaches. As rapamycin can attenuate bladder smooth muscle hypertrophy and dysfunction during the genesis of partial obstruction in vivo, we tested whether rapamycin could improve persistent function after release of obstruction (de-obstruction or REL). Female Sprague-Dawley rat bladders were partially obstructed (PBO) by suturing around both the urethra and a para-urethral steel rod, then removing the rod. One day prior to release of obstruction (preREL), voiding parameters and residual urine volume of preREL+future rapa, preREL+future veh groups were recorded. Release of obstruction (REL) was performed by suture removal following 6 weeks of PBO. For 4 more weeks after the de-obstruction, REL animals were randomized to rapamycin (REL+rapa) or vehicle (REL+veh). PBO for 6 weeks were used as positive controls. In shams, the urethra was exposed, but no suture tied. Voiding parameters and residual urine volume were measured prior to sacrifice of sham and REL+veh or REL+rapa, and PBO. Rapamycin efficacy was tested by pair-wise comparison of changes in individual voiding data from preREL+future veh or preREL+future rapa versus REL+veh or REL+rapa, respectively, as well as by comparisons of REL+veh to REL+rapa groups. Bladders were weighed and processed for a high-throughput QPCR array, and histopathology. Bladder/body mass ratios with PBO increased significantly and remained higher in the release phase in REL+veh animals. REL+rapa versus REL+veh improved residual volumes and micturition fractions toward sham levels. Three genes encoding extracellular proteins, BMP2, SOD3, and IGFBP7, correlated with functional improvement by Pearson's correlations. The promoters of these genes showed enrichment for several motifs including circadian E-boxes. While obstruction and REL augmented CLOCK and NPAS2 expression above sham levels, rapamycin treatment during release significantly blocked their expression. This experimental design of pharmaco-intervention during the de-obstruction phase revealed a novel pathway dysregulated during the clinically relevant treatment phase of obstructive bladder myopathy., (© 2020 Federation of American Societies for Experimental Biology.)

Objective: To analyze the association between neutrophil-to-lymphocyte ratio (NLR) and intravesical prostatic protrusion (IPP) in men with benign prostatic hyperplasia., Methods: Two hundred and fifty men aged >50 years who presented with lower urinary tract symptoms at our institution between 2014 and 2018 were analyzed. Pearson's method was used for analysis of the correlation between NLR and IPP. Multivariate logistic regression analysis was used to identify predictors of IPP. Further analysis according to total prostate volume (TPV) was performed., Results: The NLR correlated positively with IPP (Pearson's r = 0.459, P < 0.001) and was an independent predictor of IPP ≥10 mm (odds ratio, 2.95; 95% confidence interval, 1.59-5.47; P = 0.0006). Among the 142 men with prostates <40 cm 3 , mean NLR was 2.50 ± 0.71 in those with IPP ≥10 mm and 1.71 ± 0.57 in those with IPP < 10 mm (P < 0.001). The NLR differed significantly between those with a prostate <40 cm 3 and IPP ≥10 mm and those with a larger prostate and IPP < 10 mm (2.50 ± 0.71 vs 2.07 ± 0.77, respectively; P = 0.020)., Conclusions: NLR can be used as a surrogate marker for presence of IPP. Its clinical value would be especially important in men with a small prostate gland but high IPP. The NLR seemed to be more strongly correlated with IPP than with TPV., (© 2019 The Authors. LUTS: Lower Urinary Tract Symptoms published by John Wiley & Sons Australia, Ltd.)

We have defined a population of stem cell antigen (Sca)-1 + /CD34 + /lin - mesenchymal stem cells in the mouse urinary bladder. These cells are reduced after partial bladder outlet obstruction (PO). To test the role of Sca-1 expressed by these cells, we analyzed bladders from Sca-1 knockout (KO) mice in both uninjured male mice and male mice subjected to PO. We found that loss of Sca-1 alone had little effect on bladder development or function but reduced the total number of mesenchymal stem cells by 30%. After PO, bladders from Sca-1-null KO male mice were larger, with more collagen and less muscle, than obstructed wild-type mice. Steady-state levels of caldesmon were significantly reduced and levels of fibroblast-specific protein 1 were significantly increased in Sca-1 KO mice compared with wild-type mice after PO. In investigating the effects of PO on cell proliferation, we found that loss of Sca-1 changed the timing of cell division in CD34 + /lin - , collagen-producing, and smooth muscle cells. PO in combination with loss of Sca-1 drastically reduced the ability of CD34 + /lin - cells to form colonies in vitro. Our findings therefore support the hypothesis that Sca-1 protects the bladder from fibrotic remodeling after obstruction, in part by influencing the proliferation of cells responding to the injury.

Background: Partial bladder outlet obstruction (PBOO) promotes bladder detrusor hyperplasia, increases bladder pressure, and decreases bladder compliance. To extensively explore its underlying mechanism, our study aimed to investigate the effect of pathological hydrostatic pressure on human bladder smooth muscle cell (hBSMC) proliferation and contraction through β-adrenoceptor (ADRB) signaling in vitro., Methods: hBSMCs were subjected to pathological hydrostatic pressure (100 cm H 2 O) to investigate the effect of ADRBs on the proliferation and contraction of hBSMCs treated with its agonists and/or antagonists., Results: Firstly, exposure to 100 cm H 2 O hydrostatic pressure significantly upregulated the expression of α-smooth muscle actin (α-SMA) in hBSMCs at 6 hours, and promoted cell proliferation at 24 hours. When subjected to hydrostatic pressure alone, hBSMCs treated with ADRB2 and ADRB3 agonists for 6 hours inhibited α-SMA expression compared with untreated cells. By contrast, hBSMCs treated with ADRB2 agonists for 24 hours suppressed cell proliferation compared with untreated cells. The two classical pathways of ADRB, protein kinase A (PKA), and exchange factor directly activated by cAMP (EPAC) inhibited the contraction of hBSMCs under hydrostatic pressure via regulating mothers against decapentaplegic homolog 2 (SMAD2) activity. The proliferation of hBSMCs was mainly regulated by the EPAC pathway through extracellular signal-regulated kinase 1/2 (ERK1/2) activity., Conclusion: The contraction of hBSMCs under hydrostatic pressure was regulated by ADRB2 and ADRB3 via the PKA/EPAC-SMAD2 pathway, and the proliferation of hBSMCs was regulated by ADRB2 via the EPAC-ERK1/2 pathway. Compared with ADRB3, ADRB2 played a predominant role under pathological hydrostatic pressure. These findings markedly uncovered the underlying mechanism of ADRBs in PBOO and provided new insights into the efficient treatment of patients with PBOO., (© 2019 Wiley Periodicals, Inc.)

Purpose: To identify how the most frequently used parameters in daily clinical practice diagnosing bladder outlet obstruction (BOO) due to benign prostate hyperplasia (BPH) correlate to each other., Materials and Methods: The study included 452 patients with lower urinary tract symptoms (LUTS) of the UNICAMP urology outpatient clinic of LUTS., Inclusion Criteria: patients with BOO due to BPH who agreed to participate in the study., Exclusion Criteria: patients with urinary tract infection, neurological diseases that compromised the lower urinary tract, prior prostatic surgery, radiotherapy or urethral stenosis. Patient assessment: history, international prostate symptoms score (IPSS), nocturnal quality of life score (NQoL) questionnaires, physical and digital rectal examination (DRE), PSA, transabdominal ultrasound with intravesical prostate protrusion (IPP), post-mictional residue and free urofl owmetry., Results: There was no strong Spearman correlation among the studied variables. The only moderate correlations occurred between IPSS and NQoL (p<0001; c=0.56) and between IPP and prostate volume (p<0001; c=0.57). Weak correlations between IPP and post-mictional residue (p<0001; c=0.31) and free urofl owmetry (p<0001; c=-0.26); and between IPSS and free urofl owmetry (p<0001, c=-0.21) were observed., Conclusion: In this study, we found moderate, weak, very weak and absent correlation among the various parameters used in the diagnosis and management of BOO due to BPH. As the value of these tools is variable, the creation of a logical and objective algorithm was not possible and the treatment is based on the interpretation of clinical symptoms., Competing Interests: None declared., (Copyright® by the International Brazilian Journal of Urology.)

Congenital lower urinary-tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. About three out of 10,000 pregnancies are affected. Although several monogenic causes of functional obstruction have been defined, it is unknown whether congenital LUTO caused by anatomical blockage has a monogenic cause. Exome sequencing in a family with four affected individuals with anatomical blockage of the urethra identified a rare nonsense variant (c.2557C>T [p.Arg853 ∗ ]) in BNC2, encoding basonuclin 2, tracking with LUTO over three generations. Re-sequencing BNC2 in 697 individuals with LUTO revealed three further independent missense variants in three unrelated families. In human and mouse embryogenesis, basonuclin 2 was detected in lower urinary-tract rudiments. In zebrafish embryos, bnc2 was expressed in the pronephric duct and cloaca, analogs of the mammalian lower urinary tract. Experimental knockdown of Bnc2 in zebrafish caused pronephric-outlet obstruction and cloacal dilatation, phenocopying human congenital LUTO. Collectively, these results support the conclusion that variants in BNC2 are strongly implicated in LUTO etiology as a result of anatomical blockage., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations in leucine-rich-repeats and immunoglobulin-like-domains 2 (LRIG2) or in heparanase 2 (HPSE2) cause urofacial syndrome, a devastating autosomal recessive disease of functional bladder outlet obstruction. It has been speculated that urofacial syndrome has a neural basis, but it is unknown whether defects in urinary bladder innervation are present. We hypothesized that urofacial syndrome features a peripheral neuropathy of the bladder. Mice with homozygous targeted Lrig2 mutations had urinary defects resembling those found in urofacial syndrome. There was no anatomical blockage of the outflow tract, consistent with a functional bladder outlet obstruction. Transcriptome analysis revealed differential expression of 12 known transcripts in addition to Lrig2, including 8 with established roles in neurobiology. Mice with homozygous mutations in either Lrig2 or Hpse2 had increased nerve density within the body of the urinary bladder and decreased nerve density around the urinary outflow tract. In a sample of 155 children with chronic kidney disease and urinary symptoms, we discovered novel homozygous missense LRIG2 variants that were predicted to be pathogenic in 2 individuals with non-syndromic bladder outlet obstruction. These observations provide evidence that a peripheral neuropathy is central to the pathobiology of functional bladder outlet obstruction in urofacial syndrome, and emphasize the importance of LRIG2 and heparanase 2 for nerve patterning in the urinary tract., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Objective: Using modified sonourethrography (mSUG) with retrograde jelly injection to precisely measure the morphological characteristics of the prostatic urethra, we assessed prostatic urethral morphology associated with clinical parameters of benign prostatic hyperplasia (BPH)., Methods: BPH patients (n = 43) and control patients with localized prostate cancer (PC; n = 57) were imaged by mSUG before surgery. Using the seminal colliculus as a landmark, prostatic urethral angulation (PUA), sagittal urethral diameter, and anterior or posterior prostatic urethral length were measured. The International Prostatic Symptoms Score (IPSS) was also evaluated in all patients. The Bladder Outlet Obstruction Index (BOOI) was measured in BPH patients that could void in a pressure-flow study. Parameters were compared between BPH and PC patients, and correlations among morphological and clinical parameters were evaluated., Results: Prostatic urethras were clearly observed in all patients by mSUG. PUA, sagittal urethral diameter, and posterior urethral length were all greater in BPH than PC patients (P < .05). Among all parameters examined, PUA had the strongest correlation with IPSS (r = 0.56). Longitudinal urethral diameter showed the strongest correlation with BOOI, whereas PUA was not correlated with BOOI., Conclusions: Prostatic urethral morphology can be imaged precisely by mSUG. Morphometric measurements showed that increased PUA was strongly correlated with problematic urinary symptoms, and a flattened shape of the posterior urethra, such as extension of the sagittal urethral diameter, was correlated with urinary tract obstruction by BPH., (© 2017 John Wiley & Sons Australia, Ltd.)

Objectives: The aim of the present study was to investigate the effects of different degrees of obstruction, and the roles of inflammation, oxidative stress, and hypoxia parameters on bladder function., Methods: Thirty male Sprague-Dawley rats were divided into 3 groups (n = 10 in each group): (i) sham-operated control; (ii) severe partial bladder outlet obstruction (PBOO); and (iii) moderate PBOO. Severe and moderate PBOO were induced by urethral ligation using 3-Fr and 4-Fr catheters, respectively, for 6 weeks. After 6 weeks, the in vitro contractile responses to carbachol, electrical field stimulation, ATP and KCl were measured in bladder strips. In addition, mRNA and protein expression of nuclear factor kappa B (NF-κB) hypoxia-inducible factor (HIF) and nuclear factor, erythroid 2-like 2 (Nrf2) in bladder were determined by real-time polymerase chain reaction and western blotting. Malondialdehyde (MDA) levels in bladder tissues were also determined., Results: Rats in the severe PBOO group had the highest bladder weight. Detrusor strips from rats in the severe PBOO group exhibited 61%-82% smaller contractile responses to all four stimuli than those from the sham-operated group. Activity of NF-κB as an inflammatory marker was increased in the severe PBOO group, whereas HIF-1α and HIF-2β protein levels were increased significantly in the moderate PBOO group. A master regulator of oxidative stress, Nrf2 expression was increased in all obstructed rats. MDA levels were higher in the severe PBOO group than in sham-operated group., Conclusion: The results of the present study reveal the importance of oxidative stress-induced NF-κB signaling in bladder dysfunction with severe obstruction. Altered HIF signaling may contribute to the functional impairment after PBOO. Novel and evolving therapies targeting oxidative and/or inflammatory pathways may be a reasonable strategy for the management of lower urinary tract symptoms or benign prostatic hyperplasia., (© 2017 John Wiley & Sons Australia, Ltd.)

Anterior rectal duplication cyst is rare entity with <50 reported cases to date. It has myriad presentations like bleeding per rectum, constipation, rectal prolapsed and intestinal obstruction due to extrinsic compression of rectum. However, the association of enlarged duplication cyst compressing the bladder neck or ureter, and leading to bladder outlet obstruction or hydroureteronephrosis is extremely rare with only a handful of reported cases. We report a rare case of large anterior rectal duplication cyst in a young girl leading to acute urinary retention with bladder outlet obstruction which was eventually managed by laparoscopic-assisted transabdominal surgical excision of the cyst. The authors believe that such an association has not been previously reported in this age group., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

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-β signaling pathway by inhibiting the expression of TGFβR1. Then, we conclude microRNA-101b is induced by hypoxia and represses fibrosis of BSMCs by inhibiting the expression of TGFβR1 through TGF-β signaling pathway, and it may be an anti-fibrotic miRNA for therapy. © 2018 IUBMB Life, 71(1):235-243, 2019., (© 2018 International Union of Biochemistry and Molecular Biology.)

Aims: To investigate the effects of diets on factors and markers of inflammation and fibrosis in unobstructed and obstructed bladders of male Wistar rats., Materials and Methods: Partial BOO was surgically induced in twelve-week old rats after feeding on different diets for eight (8) weeks. Feeding continued for 4 weeks after surgery. Rats were divided into sham-operated and BOO groups as follow: control, high-carbohydrate (HCD), high-fat (HFD) and high-protein (HPD). After the feeding period, bladder weight, CRP, nerve growth factor (NGF), tissue growth factor-β (TGF-β), connective tissue growth factor (CTGF), hypoxia inducible factor-1α (HIF-1 α), platelet-derived growth factor-A (PDGF-A) and CXCL12 were all determined., Key Findings: In both unobstructed and obstructed bladders, CRP was increased in animals fed on the HFD (P < 0.05). NGF was increased in animals fed on HFD and HPD but decreased only in HCD-BOO. CXCL12 was increased in animals fed on HFD and HPD (P < 0.05) and decreased in HCD. The HCD-BOO group exhibited a decrease in CXCL12, while CXCL12 increased in HFD-BOO. TGF-β was elevated in HFD and all the dietary-BOO groups, but animals with obstructed bladders fed on the HPD and HCD had significant reduction in TGF-β expression. CTGF was increased in HFD- and HPD-fed animals. HIF-1α, PDGF-A and collagen were increased in both HFD dietary groups and HPD-BOO., Significance: Feeding on a high fat diet results in increased activity of factors and mediators of inflammation and fibrosis in both unobstructed and obstructed rat bladders. This might increase predisposition to or further worsen symptoms in BOO., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aims: Open surgery is the most commonly used methodological approach for generating a partial bladder outlet obstruction (pBOO) animal model. Surgical suturing closing a part of the urethral meatus induces comparable pathophysiological changes in bladder and renal functions, but the optimum degree of obstruction that closely mimics the clinical pathology of pBOO has not been elucidated. We investigated the optimum obstruction level by performing a comprehensive time-dependent analysis of the stability and reliability of this novel animal model., Methods: Six- to eight-week-old female BALB/c mice were divided into three groups according to the degree of urethral meatus stricture (UMS). Non-operated mice served as controls, and a pBOO model generated using the traditional method served as a positive control. A cystometric evaluation and long-term studies were performed to evaluate the validity and reliability of this novel animal model. An additional 35 mice were used to investigate the protein expression levels and histopathological features 24 h and 14 days postoperatively, respectively., Results: The characteristic cystometry features in the UMS group revealed increased changes in pressure-related parameters compared with the control. The 1/3 UMS model is an optional pBOO animal model because the cystometric evaluation and histopathological studies revealed a striking resemblance between the 1/3 UMS model and the model generated using the traditional open-surgery method., Conclusions: The minimally invasive UMS model required less time and produced minimal alterations in pathophysiologically relevant processes compared with the traditional surgery model. Suturing to cause UMS produced effective and repeatable patterns in bladder function investigations in mice., (© 2018 The Authors. Neurourology and Urodynamics Published by Wiley Periodicals, Inc.)

Aims: To evaluate the therapeutic effect of TAC-302, a cyclohexenoic fatty alcohol derivative, on bladder denervation-related storage and voiding dysfunctions in rats with bladder outlet obstruction (BOO)., Methods: BOO was achieved by partial ligature of the proximal urethra in female rats. Two weeks later, BOO rats were divided into two groups and treated orally with vehicle or 10 mg/kg TAC-302 twice a day for 4 weeks. Urodynamic and immunohistochemical evaluation of the bladder muscle layer was performed. In another study, the BOO rats were treated with intravenous tamsulosin at cystometry. The detrusor contractility in each group was evaluated using the modified Shafer's nomogram., Results: Two weeks after BOO, the rats showed significant increases in non-voiding contraction (NVCs) and residual urine volume (RUV) compared to the sham group. Moreover, 6 weeks after BOO, BOO vehicle rats showed significant increases in NVCs and RUV and decreases in detrusor contractility and in the nerve fiber density in the urinary bladder compared to the sham group. BOO-induced denervation of the urinary bladder was partially improved by oral treatment with TAC-302. Oral treatment with TAC-302 significantly reduced the amplitude and frequency of NVCs (P < 0.05) and increased detrusor contractility and tended to reduce RUV compared with the BOO vehicle group. In contrast, the intravenous administration of tamsulosin significantly reduced the frequency of NVCs, but not RUV., Conclusions: TAC-302 improved storage and voiding dysfunctions by improving bladder denervation and detrusor underactivity even when the treatment was started after storage and voiding dysfunctions had already occurred., (© 2018 Wiley Periodicals, Inc.)

Objective: To investigate the anti-oxidative stress and preventive effect of modified Gongjin-dan (WSY-1075) in a detrusor underactivity rat model., Methods: Rats were randomly allocated to three groups: shamoperated (control), bladder outlet obstruction-induced detrusor underactivity (BOO-DU), and BOO-DU with WSY-1075 (WSY) groups. WSY-1075 was orally administrated to rats 200 mg daily for 2 weeks prior to the operation and 4 weeks after the operation. Bladder outlet obstruction was surgically induced in rats by ligation around the urethra avoiding total obstruction. Cystometrography was conducted on rats in each group for examination of bladders., Results: Compared with the control group, bladder outlet obstruction led to a significant increase in oxidative stress with consequent changes to molecular composition, and decrease in maximal detrusor pressure (P<0.05). WSY-1075 treatment significantly suppressed oxidative stress and prevented degenerative and dysfunctional changes in bladder, as compared with BOO-DU group (P<0.05)., Conclusion: WSY-1075 had beneficial effect on prevention of BOO-DU.

Kennedy's disease, or spinal and bulbar muscular atrophy (SBMA), is an X-linked neuromuscular condition clinically characterised by weakness, atrophy and fasciculations of the limb and bulbar muscles, as a result of lower motor neuron degeneration. The disease is caused by an abnormally expanded triplet repeat expansions in the ubiquitously expressed androgen receptor gene, through mechanisms which are not entirely elucidated. Over the years studies from both humans and animal models have highlighted the involvement of cell populations other than motor neurons in SBMA, widening the disease phenotype. The most compelling aspect of these findings is their potential for therapeutic impact: muscle, for example, which is primarily affected in the disease, has been recently shown to represent a valid alternative target for therapy to motor neurons. In this review, we discuss the emerging study of the extra-motor neuron involvement in SBMA, which, besides increasingly pointing towards a multidisciplinary approach for affected patients, deepens our understanding of the pathogenic mechanisms and holds potential for providing new therapeutic targets for this disease., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Bladder outlet obstruction (BOO) and the ensuing clinical lower urinary tract dysfunction are common in elderly patients. BOO is accompanied by urodynamic changes in bladder function and leads to organ fibrosis and ultimately loss of contractility. Comprehensive transcriptome analysis of bladder samples from human patients with different urodynamically defined phenotypes of BOO revealed tumor necrosis factor (TNF)-α as the top upstream signaling pathway regulator. Herein, we validated next-generation sequencing and pathway analysis in cell-based models using bladder smooth muscle and urothelial cells exposed to TNF-α. miRNA profiling and transcriptome analysis of TNF-α-treated bladder smooth muscle cells revealed striking similarities with human BOO. Using a comparative approach, TNF-specific and TNF-independent pathways were delineated in human biopsy specimens. Concomitant down-regulation of smooth muscle cell-specific miRNAs and smooth muscle markers after TNF-α treatment was in accordance with the loss of contractility in humans in advanced obstruction-induced bladder remodeling. The expression levels of four abundant TNF-regulated miRNAs were modulated; the compensatory up-regulation of miR-199a-5p reduced NF-κB signaling. Essential hubs of TNF-α signaling pathways mitogen-activated protein kinase kinase kinase (apoptosis signal-regulating kinase 1) and inhibitor of nuclear factor κ B kinase subunit β (IκB kinase β) were targeted by miR-199a-5p. miR-199a-5p might be part of a negative feedback loop, reducing the impact of TNF, whereas its down-regulation in acontractile bladders from BOO patients advances the disease. The compensatory up-regulation of miR-199a-5p together with TNF-α inhibition may be therapeutically beneficial., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: To investigate the protective effect of epigallocatechin gallate (EGCG), a green tea extract, and its underlying mechanism on bladder dysfunction in a rat model of bladder outlet obstruction (BOO)., Materials and Methods: Sprague-Dawley rats of BOO were surgically induced and followed by treatment with EGCG (5 mg/kg/day) or saline (control) via intraperitoneal injection. Cystometry was performed on four weeks postoperatively in conscious rats. H&E, Masson trichrome, and TUNEL staining were performed to observe tissue alterations. Oxidative stress markers were measured, and protein expression of Nrf2-ARE pathway was examined by immunohistochemistry and Western blotting., Results: Our data showed that EGCG could increase the peak voiding pressure and bladder compliance and prolong micturition interval of BOO rats compared with control and finally reduce the frequency of urinary. EGCG could ameliorate the increase of collagen fibers and ROS induced by obstruction and increase the activity of SOD, GSH-Px, and CAT. The level of cell apoptosis was decreased in BOO rats treated with EGCG compared with control, and caspase-3 expression was reduced as well. Moreover, EGCG could activate the Nrf2 expression with elevation of its target antioxidant proteins., Conclusions: EGCG alleviates BOO-induced bladder dysfunction via suppression of oxidative stress and activation of the protein expression of Nrf2-ARE pathway.

Background: Benign prostatic hyperplasia (BPH) is a common urologic disease in older men. Prostatic inflammation research has focused on the magnitude of inflammation; its location has received little attention. We investigated whether the anatomic location of prostatic inflammation is related to the severity of lower urinary tract symptoms (LUTS), measured subjectively and objectively., Methods: We retrospectively analyzed hematoxylin+eosin-stained tissue specimens from 179 BPH patients who underwent transurethral resection of the prostate (TURP) or holmium laser enucleation of the prostate (HoLEP). Chronic prostatic inflammation was assessed by the grade (lymphocyte density), extent (lymphocyte distribution), and location of inflammation. Each inflammation-finding type was evaluated in relation to these clinical parameters: age, prostate volume, prostate-specific antigen (PSA) value, body mass index (BMI), the frequency of acute urinary retention (AUR) episodes, the international prostatic symptom score (IPSS), and urodynamic study results., Results: The magnitude and extent of inflammation were not associated with any clinical parameters. We classified the BPH patients into stromal (n = 72) versus non-stromal (n = 105) groups based on their inflammation's dominant location. The stromal group's prostatic volume was significantly larger than the non-stromal group's (63.8 vs 53.8 mL; P = 0.032). AUR episodes were more significantly frequent in the stromal group (36.1% vs 11.4%; P = 0.006). Between-group differences in storage parameters (ie, maximum cystometric capacity) in the urodynamic study were not significantly different. Voiding parameters differed significantly between the stromal and non-stromal groups: maximum detrusor pressure (maxPdet) (116.8 vs 94.5 cmH 2 O, P = 0.014), Pdet at the maximum flow rate (Qmax) (95.8 vs 75.4 cmH 2 O, P = 0.014), and the bladder outlet obstruction index (BOOI) (78.5 vs 56.3, P = 0.014). The stromal group's Qmax was significantly lower than the non-stromal group's (7.3 vs 9.8 mL/s, P = 0.004)., Conclusions: The location of inflammation in the prostate might be an important factor affecting the severity of LUTS, especially voiding dysfunction., (© 2018 Wiley Periodicals, Inc.)