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

The interstitial cells in bladder lamina propria (LP-ICs) are believed to be involved in sensing/afferent signaling in bladder mucosa. Transient receptor potential (TRP) cation channels act as mechano- or chemo-sensors and may underlie some of the sensing function of bladder LP-ICs. We aimed to investigate the molecular and functional expression of TRP channels implicated in bladder sensory function and Piezo1/Piezo2 channels in cultured LP-ICs of the human bladder. Bladder tissues were obtained from patients undergoing cystectomy. LP-ICs were isolated and cultured, and used for real-time reverse transcription-quantitative polymerase chain reaction, immunocytochemistry, and calcium-imaging experiments. At the mRNA level, TRPA1, TRPV2, and Piezo1 were expressed most abundantly. Immunocytochemical staining showed protein expression of TRPA1, TRPV1, TRPV2, TRPV4, TRPM8, as well as Piezo1 and Piezo2. Calcium imaging using channel agonists/antagonists provided evidence for functional expression of TRPA1, TRPV2, TRPV4, Piezo1, but not of TRPV1 or TRPM8. Activation of these channels with their agonist resulted in release of adenosine triphosphate (ATP) from LP-ICs. Inhibition of TRPV2, TRPV4 and Piezo1 blocked the stretch induced intracellular Ca2+ increase. Whereas inhibition of TRPA1 blocked H2O2 evoked response in LP-ICs. Our results suggest LP-ICs of the bladder can perceive stretch or chemical stimuli via activation of TRPV2, TRPV4, Piezo1 and TRPA1 channels. LP-ICs may work together with urothelial cells for perception and transduction of mechanical or chemical signals in human-bladder mucosa. [ABSTRACT FROM AUTHOR]

Urothelial cells have been implicated in bladder mechanosensory transduction, and thus, initiation of the micturition reflex. Cell deformation caused by tension forces at an air-liquid interface (ALI) can induce an increase in intracellular Ca2+ concentration ([Ca2+]i) and ATP release in some epithelial cells. In this study, we aimed to examine the cellular mechanisms underlying ALI-induced [Ca2+]i increase in cultured urothelial cells. The ALI was created by stopping the influx of the perfusion but maintaining efflux. The [Ca2+]i increase was measured using the Ca2+ imaging method. The ALI evoked a reversible [Ca2+]i increase and ATP release in urothelial cells, which was almost abolished by GdCl3. The specific antagonist of the transient receptor potential vanilloid (TRPV4) channel (HC0674) and the antagonist of the pannexin 1 channel (10panx) both diminished the [Ca2+]i increase. The blocker of Ca2+-ATPase pumps on the endoplasmic reticulum (thapsigargin), the IP3 receptor antagonist (Xest-C), and the ryanodine receptor antagonist (ryanodine) all attenuated the [Ca2+]i increase. Degrading extracellular ATP with apyrase or blocking ATP receptors (P2X or P2Y) with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) significantly attenuated the [Ca2+]i increase. Our results suggest that both Ca2+ influx via TRPV4 or pannexin 1 and Ca2+ release from intracellular Ca2+ stores via IP3 or ryanodine receptors contribute to the mechanical responses of urothelial cells. The release of ATP further enhances the [Ca2+]i increase by activating P2X and P2Y receptors via autocrine or paracrine mechanisms. [ABSTRACT FROM AUTHOR]

Background: Penile hypersensitivity is one of the main pathological mechanisms of premature ejaculation. However, little is known about the neurophysiological mechanism of penile peripheral nerve sensitization. Piezo Type Mechanosensitive Ion Channel Component 2 (PIEZO2), was recently identified as a mechanically sensitive channel. Objectives: This study explored the possible neural mechanisms of PIEZO2 action in the mechanisms of premature ejaculation using molecular biology and electrophysiology approaches. Materials and methods: One hundred seventy male rats and 85 female rats were recruited. The females were induced estrus by injection of estradiol benzoate and progesterone followed by surgically castrated. Subsequently, the copulatory behaviors were record by a video camera six times, once a week. The last three mating processes of 134 male rats were successfully recorded. The males were divided into three groups according to ejaculation frequency value. Immunocytochemical and molecular methods as well as whole‐cell patch clamp recording were used to show the difference between premature ejaculation rats and control rats. To further clarify the involvement of PIEZO2 in premature ejaculation, we constructed a PIEZO2 knockdown model in rats by intrathecal injection of PIEZO2 antisense oligodeoxynucleotides. Results: We showed that PIEZO2 in the penis head and in the dorsal root ganglia(DRG) were significantly increased in premature ejaculation rats. Whole‐cell patch clamp recording demonstrated that mechanical stimulation evoked a higher inward current density in premature ejaculation rats compared with control rats, which could be inhibited by the PIEZO2‐specific antagonist, FM1‐43. PIEZO2 knockdown experiments revealed that the inward current density induced by mechanical stimulation was significantly decreased in PIEZO2 knockdown rats, and that the mount frequency and ejaculation latency and frequency were significantly improved in PIEZO2 knockdown rats. Discussion and Conclusion: Our data demonstrate PIEZO2 involvement in peripheral nerve sensitization, indicating that pharmacological antagonism of PIEZO2 may be a useful strategy for treating premature ejaculation. [ABSTRACT FROM AUTHOR]

Deteriorating urban water quality has attracted considerable attention in China. We investigated the contamination levels and distribution of heavy metals (As, Cd, Cu, Ni, Pb, and Zn) in Yuxi River water and sediments, and assessed the heavy metal accumulation capability of five species of submerged macrophytes: Vallisneria natans (Lour.) Hara, Potamogeton pectinatus L., Hydrilla verticillata (L. f.) Royle, Myriophyllum spicatum L., and Potamogeton crispus L. Samples were collected from upstream and downstream locations in different season. The results showed that the levels of heavy metals in the downstream areas were higher than in the upstream areas. Heavy metal concentrations in the river water during the dry seasons were higher than those during the rainy seasons, and the opposite results appeared in sediments and submerged macrophytes. In general, the river was slightly contaminated by heavy metals, and the concentrations of Pb and Ni in this river should serve as a warning, while Cd and Zn pollution in the sediments desperately needs to be removed. Furthermore, Potamogeton pectinatus L. showed a higher accumulation capacity for these metals among the five native submerged macrophytes and could be defined as a hyperaccumulator for Cd. Therefore, the potential use of native aquatic plants in contaminated rivers is worth further exploration. [ABSTRACT FROM AUTHOR]

• Bladder primary sensory neurons are divided into phasic and tonic neurons based on the responses to super-threshold stimuli. • Phasic and tonic neurons have distinct electrical properties. • No age-related change in excitability was found in tonic bladder sensory neurons. • An age-related reduction in excitability was observed in phasic bladder sensory neurons. Previous studies have revealed an impairment in bladder sensory transduction in aged animals. To examine the contributions of electrical property changes of bladder primary afferents to this impairment, we compared the electrical properties of dorsal root ganglion (DRG) neurons innervating the bladder among young (3 months), middle-aged (12 months), and old (24 months) female rats. The DRG neurons were labeled using axonal tracing techniques. Whole-cell current-clamp recordings of small and medium-sized neurons were performed to assess their passive and active properties. Two patterns of firing were identified based on responses to super-threshold stimuli (1.5, 2.0, 2.5, and 3.0 × rheobase): tonic neurons fired more action potentials (APs), whereas phasic neurons fired only one AP at the onset of stimulus. Tonic neurons were smaller and had a slower rate of AP rise, longer AP duration, more depolarized voltage threshold, and greater rheobase than phasic neurons. In phasic neurons, there was an age-associated increase in voltage threshold and an increase of rheobase (P < 0.05), suggesting an age-related decrease in excitability. In addition, both middle-aged and old rats had longer AP durations and slower rates of AP rise than young rats (P < 0.05). In tonic neurons, old rats had a greater AP overshoot and greater rate of AP rise, but no age-associated changes were identified in any other electrical properties. Our results suggest that the electrical properties of tonic and phasic bladder afferents are differentially altered with aging. A decrease in excitability may contribute to age-related reductions in bladder sensory function. [ABSTRACT FROM AUTHOR]

Background and Aims Kidney injury molecule-1 (KIM-1) was identified the most highly upregulated protein in chronic kidney diseases and prolonged KIM-1 expression may be maladaptive. The present study was aimed to investigate urinary, renal and plasma KIM-1 levels and to analyze association between KIM-1 levels with clinical and pathological indexes in adult Henoch-Schönlein purpura (HSP) patients. Methods Twenty healthy individuals, 20 HSP patients without nephritis and 35 HSP patients with nephritis were recruited. Urinary and plasma KIM-1 levels were determined by ELISA and Luminex, respectively. Renal KIM-1 expression was evaluated by immunohistochemistry. Results HSP patients with nephritis were characterized as elevated levels of urinary, renal and plasma KIM-1. Those with more severe tubular injury of renal biopsy tissues presented significantly higher urinary and renal KIM-1 levels compared to control and patients without nephritis. Urinary and renal levels of KIM-1 were positively correlated with blood urea nitrogen and proteinuria, while they were negatively correlated with eGFR at both baseline and after two years follow-up. Moreover, plasma KIM-1 levels were associated with blood urea nitrogen and proteinuria as well. Further univariate correlation analysis indicated urinary and renal KIM-1 levels were positively correlated with interstitial inflammation index and tubulointerstitial chronicity index. Only urinary KIM-1 levels were associated with interstitial inflammation index, tubulointerstitial chronicity index and extracapillary glomerular activity index, after logistic regression analysis. The area under the curve (AUC) for urinary KIM-1/Cr predicting progression of renal damage was significantly greater than the AUC for proteinuria. Conclusions This finding suggests that measurement of urinary and renal KIM-1 level may be helpful to evaluate severity of renal pathological damage and prognosis in adult HSP patients with nephritis. [ABSTRACT FROM AUTHOR]

We aimed to investigate the expression and motor modulatory roles of several mechano-sensitive channels (MSCs) in human ureter. Human proximal ureters were obtained from eighty patients subjected to nephrectomy. Expression of MSCs at mRNA, protein and functional levels were examined. Contractions of longitudinal ureter strips were recorded in organ bath. A fluorescent probe Diaminofluoresceins was used to measure nitric oxide (NO). RT-PCR analyses revealed predominant expression of Piezo1 and TRPV2 mRNA in intact ureter and mucosa. Immunofluorescence assays indicate proteins of MSCs (Piezo1/Piezo2, TRPV2 and TRPV4) were mainly distributed in the urothelium. Ca2+ imaging confirmed functional expression of TRPV2, TRPV4 and Piezo1 in cultured urothelial cells. Specific agonists of Piezo1 (Yoda1, 3-300 μM) and TRPV2 (cannabidiol, 3-300 μM) attenuated the frequency of ureteral contractions in a dose-dependent manner while the TRPV4 agonist GSK1016790A (100 nM-1 μM) exerted no effect. The inhibitory effects of Piezo1 and TRPV2 agonists were significantly blocked by the selective antagonists (Dooku 1 for Piezo1, Tranilast for TRPV2), removal of the mucosa, and pretreatment with NO synthase inhibitor L-NAME (10 μM). Yoda1 (30 μM) and cannabidiol (50 μM) increased production of NO in cultured urothelial cells. Our results suggest that activation of Piezo1 or TRPV2 evokes NO production and release from mucosa that may mediate mechanical stimulus-induced reduction of ureter contractions. Our findings support the idea that targeting Piezo1 and TRPV2 channels may be a promising pharmacological strategy for ureter stone passage or colic pain relief., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Liu, Wang, Wang, Ding, Liu, Liu, Wen, Sun, Zu, Zhang and Yan.)

Urothelial cells have been implicated in bladder mechanosensory transduction, and thus, initiation of the micturition reflex. Cell deformation caused by tension forces at an air-liquid interface (ALI) can induce an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) and ATP release in some epithelial cells. In this study, we aimed to examine the cellular mechanisms underlying ALI-induced [Ca 2+ ] i increase in cultured urothelial cells. The ALI was created by stopping the influx of the perfusion but maintaining efflux. The [Ca 2+ ] i increase was measured using the Ca 2+ imaging method. The ALI evoked a reversible [Ca 2+ ] i increase and ATP release in urothelial cells, which was almost abolished by GdCl 3 . The specific antagonist of the transient receptor potential vanilloid (TRPV4) channel (HC0674) and the antagonist of the pannexin 1 channel ( 10 panx) both diminished the [Ca 2+ ] i increase. The blocker of Ca 2+ -ATPase pumps on the endoplasmic reticulum (thapsigargin), the IP3 receptor antagonist (Xest-C), and the ryanodine receptor antagonist (ryanodine) all attenuated the [Ca 2+ ] i increase. Degrading extracellular ATP with apyrase or blocking ATP receptors (P2X or P2Y) with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) significantly attenuated the [Ca 2+ ] i increase. Our results suggest that both Ca 2+ influx via TRPV4 or pannexin 1 and Ca 2+ release from intracellular Ca 2+ stores via IP3 or ryanodine receptors contribute to the mechanical responses of urothelial cells. The release of ATP further enhances the [Ca 2+ ] i increase by activating P2X and P2Y receptors via autocrine or paracrine mechanisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wen, Chen, Zhao, Zu, Zhao, Wang and Zhang.)

Literature documents an age-related reduction of bladder sensory function. Transient receptor potential vanilloid (TRPV)1 or TRPV4 channels have been implicated in bladder mechanotransduction. To investigate contributions of TRPV1 or TRPV4 to the age-related reduction of bladder sensory function, bladder responses to capsaicin (CAP; TRPV1 agonist) and GSK-1016790A (GSK; TRPV4 agonist) in retired breeder (RB; 12-15 mo) and young adult (2-3 mo) female rats were compared using multiple methods. Metabolic cage and continuous infusion cystometry [cystometrogram (CMG)] recordings revealed that RB rats exhibit larger bladder capacity and lower voiding frequency. RB rats also have a greater intravesical pressure threshold for micturition; however, the voiding contraction strength was equivalent to that in young rats. CAP (1 μM) or GSK (20 nM) administered intravesically evoked smaller changes in all CMG parameters in RB rats. In vitro, CAP (1 μM) or GSK (20 nM) evoked smaller enhancement of bladder strip contractions, while the muscarinic receptor agonist carbachol (at 100, 300, and 1,000 nM) elicited greater amplitude contractions in RB rats. Patch-clamp recording revealed smaller CAP (100 nM) induced inward currents in bladder primary sensory neurons, and Ca 2+ imaging revealed smaller GSK (20 nM) evoked increases in intracellular Ca 2+ concentration in urothelial cells in RB rats. These results suggest that RB rats have a decreased bladder sensory function commonly observed in elderly women, and could be used as an animal model to study the underling mechanisms. Reduced functional expression of TRPV1 in bladder afferents or reduced functional expression of urothelial TRPV4 may be associated with the diminished sensory function.

Objectives: Paris polyphylla var. yunnanensis (PPVY), a Chinese herb, has long been used for cancer treatment, and its steroidal saponins are suggested to exert an anti-tumor activity, however, the underlying mechanism is incompletely understood and their effect on bladder cancer (BC) remains unknown. The present study is thus designed to address these issues., Material and Methods: Total steroidal saponins were extracted with ethanol from PPVY and used to treat BC cells (HT1197 and J82 carrying mutant p53). Gene expression was determined using qPCR and immunoblotting and cell cycle analyzed using flow cytometry. DNA damage response activation was assessed using immunofluorescence staining., Results: PPVY saponins treatment led to dose-dependent declines in the number of both HT1197 and J82 cells with IC50 approximately 1.2 μg/ml, which was coupled with strong growth arrest at G2/M phase and the activation of DNA damage response pathway. Moreover, the clonogenic potential of these cells was severely impaired even in the presence of low concentrations of PPVY saponins. Mechanistically, PPVY saponins induced the degradation of mutant p53 while stimulated CDKN1A gene transcription. Phosphorylated AKT was diminished in PPVY saponin-treated cells, but its specific inhibitor LY294002 exhibited significantly weaker efficacy in inducing CDKN1A expression than did PPVY saponins., Conclusion: PPVY saponins activate DNA damage response pathway, degrade mutant p53 and stimulate CDKN1A expression, thereby inhibiting BC cell growth. Given their poor absorption via oral administration, PPVY saponins may be applicable for intravesical instillations in BC treatment.

Objective: To evaluate the potential anti-prostate cancer effects of Paris polyphylla ethanol extract (PPEE) and its underlying mechanisms., Materials and Methods: The anti-proliferation activity of PPEE was tested on PC3 and DU145 cells using Cell Counting Kit-8 assay. The pro-apoptotic and cell cycle arrest effects of PPEE were confirmed by flow cytometry. Apoptosis of prostate cancer cells was induced by PPEE through endogenous and exogenous pathways. A mouse xenograft model was used to examine its anti-prostate cancer effects in vivo ., Results: We found that the IC50 of PPEE on PC3 cells was 3.98 µg/ml and the IC50 of PPEE on DU145 cells was 8 µg/ml. PPEE induced prostate cancer cell apoptosis in a concentration dependent manner, through endogenous and exogenous pathways. PPEE induced PC3 cell cycle arrest in G0/G1 and G2/M phases, while in DU145cell it induced cell arrest in the G0/G1 phase. PPEE inhibited the growth of prostate cancer cells in vivo ., Conclusion: PPEE could inhibit prostate cancer growth in vitro and in vivo , induce apoptosis of prostate cancer cells, and cause cell cycle arrest, which laid the foundation for further research on the anti-tumor mechanism of PPEE.