Satoru Kira

Functional Role for Piezo1 in Stretch-evoked Ca2+ Influx and ATP Release in Urothelial Cell Cultures

Background: The Piezo1 channel was recently identified as a genuine mechanosensor in mammalian cells. Results: Urothelial cells exhibited a Piezo1-dependent increase in cytosolic Ca2+ concentrations in response to mechanical stretch stimuli, leading to ATP release. Conclusion: Piezo1 senses extension of the bladder urothelium, which is converted into an ATP signal. Significance: Inhibition of Piezo1 might provide a new treatment for bladder dysfunction. The urothelium is a sensory structure that contributes to mechanosensation in the urinary bladder. Here, we provide evidence for a critical role for the Piezo1 channel, a newly identified mechanosensory molecule, in the mouse bladder urothelium. We performed a systematic analysis of the molecular and functional expression of Piezo1 channels in the urothelium. Immunofluorescence examination demonstrated abundant expression of Piezo1 in the mouse and human urothelium. Urothelial cells isolated from mice exhibited a Piezo1-dependent increase in cytosolic Ca2+ concentrations in response to mechanical stretch stimuli, leading to potent ATP release; this response was suppressed in Piezo1-knockdown cells. In addition, Piezo1 and TRPV4 distinguished different intensities of mechanical stimulus. Moreover, GsMTx4, an inhibitor of stretch-activated channels, attenuated the Ca2+ influx into urothelial cells and decreased ATP release from them upon stretch stimulation. These results suggest that Piezo1 senses extension of the bladder urothelium, leading to production of an ATP signal. Thus, inhibition of Piezo1 might provide a promising means of treating bladder dysfunction.

Functional roles of TRPV1 and TRPV4 in control of lower urinary tract activity: dual analysis of behavior and reflex during the micturition cycle

The present study used a dual analysis of voiding behavior and reflex micturition to examine lower urinary tract function in transient receptor potential vanilloid (TRPV)1 knockout (KO) mice and TRPV4 KO mice. In metabolic cage experiments conducted under conscious conditions (i.e., voluntary voiding behavior), TRPV4 KO mice showed a markedly higher voiding frequency (VF; 19.3 ± 1.2 times/day) and a smaller urine volume/voiding (UVV; 114 ± 9 μl) compared with wild-type (WT) littermates (VF: 5.2 ± 0.5 times/day and UVV: 380 ± 34 μl). Meanwhile, TRPV1 KO mice showed a similar VF to WT littermates (6.8 ± 0.5 times/day) with a significantly smaller UVV (276 ± 20 μl). Water intake among these genotypes was the same, but TRPV4 KO mice had a larger urine output than the other two groups. In cystometrogram experiments conducted in decerebrate unanesthetized mice (i.e., reflex micturition response), no differences between the three groups were found in any cystometrogram variables, including voided volume, volume threshold for inducing micturition contraction, maximal voiding pressure, and bladder compliance. However, both TRPV1 KO and TRPV4 KO mice showed a significant number of nonvoiding bladder contractions (NVCs; 3.5 ± 0.9 and 2.8 ± 0.7 contractions, respectively) before each voiding, whereas WT mice showed virtually no NVCs. These results suggest that in the reflex micturition circuit, a lack of either channel is involved in NVCs during bladder filling, whereas in the forebrain, it is involved in the early timing of urine release, possibly in the conscious response to the bladder instability.

Purinergic control of AMPK activation by ATP released through connexin 43 hemichannels - pivotal roles in hemichannel-mediated cell injury.

ABSTRACT Connexin hemichannels regulate many cell functions. However, the molecular mechanisms involved remain elusive. Hemichannel opening causes loss of ATP, we therefore speculated a potential role for AMPK in the biological actions of hemichannels. Activation of hemichannels by removal of extracellular Ca2+ led to an efflux of ATP and a weak activation of AMPK. Unexpectedly, dysfunction of hemichannels markedly potentiated AMPK activation, which was reproduced by promotion of extracellular ATP degradation or inhibition of P2 purinoceptors but counteracted by exogenous ATP. Further analysis revealed that ATP induced a purinoceptor-dependent activation of Akt and mTOR. Suppression of Akt or mTOR augmented AMPK activation, whereas activation of Akt by transfection of cells with myristoylated Akt, a constitutively active form of Akt, abolished AMPK activation. In a pathological model of hemichannel opening triggered by Cd2+, disclosure of hemichannels similarly enhanced AMPK activity, which protected cells from Cd2+-induced cell injury through suppression of mTOR. In summary, our data point to a channel-mediated mechanism for the regulation of AMPK through a purinergic signaling pathway. Furthermore, we define AMPK as a pivotal molecule that underlies the regulatory effects of hemichannels on cell survival.

Urothelial ATP exocytosis: Regulation of bladder compliance in the urine storage phase

The bladder urothelium is more than just a barrier. When the bladder is distended, the urothelium functions as a sensor to initiate the voiding reflex, during which it releases ATP via multiple mechanisms. However, the mechanisms underlying this ATP release in response to the various stretch stimuli caused by bladder filling remain largely unknown. Therefore, the aim of this study was to elucidate these mechanisms. By comparing vesicular nucleotide transporter (VNUT)-deficient and wild-type male mice, we showed that ATP has a crucial role in urine storage through exocytosis via a VNUT-dependent mechanism. VNUT was abundantly expressed in the bladder urothelium, and when the urothelium was weakly stimulated (i.e. in the early filling stages), it released ATP by exocytosis. VNUT-deficient mice showed reduced bladder compliance from the early storage phase and displayed frequent urination in inappropriate places without a change in voiding function. We conclude that urothelial, VNUT-dependent ATP exocytosis is involved in urine storage mechanisms that promote the relaxation of the bladder during the early stages of filling.

Decreased Expression of the Epithelial Ca2+ Channel TRPV5 and TRPV6 in Human Renal Cell Carcinoma Associated With Vitamin D Receptor

PURPOSE We investigated the expression of epithelial Ca(2+) channel TRPV (transient receptor potential vanilloid subfamily) 5 and 6, and vitamin D receptor in primary human renal cell carcinoma and benign peritumor tissues, and assessed the possible association between TRPV5/6 and vitamin D receptor expression. MATERIALS AND METHODS Fresh-frozen primary tumor and peritumor tissues from 27 patients diagnosed with renal cell carcinoma were analyzed for TRPV5/6 and vitamin D receptor expression by quantitative reverse transcriptase-polymerase chain reaction, Western blot and immunohistochemistry. RESULTS Quantitative reverse transcriptase-polymerase chain reaction revealed that TRPV5/6 and vitamin D receptor expression was decreased 38.11, 4.44 and 3.20 times in renal cell carcinoma vs normal kidney tissue (p = 0.012, 0.002 and 0.020, respectively). Relatively higher expression was noted for chromophobe renal cell carcinoma than for the other renal cell carcinoma subtypes. Vitamin D receptor mRNA expression significantly correlated with that of TRPV6 (r = 0.508, p = 0.007) and TRPV5 (r = 0.697, p = 0.032) in renal cell carcinoma. Western blot showed results similar to those of reverse transcriptase-polymerase chain reaction. Different expression was detected between kidney and renal cell carcinoma tissue. Immunohistochemical analysis verified strong detection of TRPV5/6 and vitamin D receptor in distal nephrons but demonstrated weak or no immunostaining much more often in renal cell carcinoma. CONCLUSIONS Decreased TRPV5/V6 expression was noted in renal cell carcinoma, which correlated with vitamin D receptor. Different expression was also detected among the different renal cell carcinoma histopathological subtypes. Our observations suggest that altered vitamin D receptor expression may be associated with renal cell carcinoma carcinogenesis via TRPV5/6.

Clock Genes Regulate the Circadian Expression of Piezo1, TRPV4, Connexin26, and VNUT in an Ex Vivo Mouse Bladder Mucosa

Objectives ClockΔ19/Δ19 mice is an experimental model mouse for nocturia (NOC). Using the bladder mucosa obtained from ClockΔ19/Δ19 mice, we investigated the gene expression rhythms of mechanosensory cation channels such as transient receptor potential cation channel subfamily V member 4 (TRPV4) and Piezo1, and main ATP release pathways including vesicular nucleotide transporter (VNUT) and Connexin26(Cx26), in addition to clock genes. Materials and methods Eight- to twelve-week-old male C57BL/6 mice (WT) and age- and sex-matched C57BL/6 ClockΔ19/Δ19 mice, which were bred under 12-h light/dark conditions for 2 weeks, were used. Gene expression rhythms and transcriptional regulation mechanisms in clock genes, mechanosensor, Cx26 and VNUT were measured in the mouse bladder mucosa, collected every 4 hours from WT and ClockΔ19/Δ19 mice using quantitative RT-PCR, a Western blot analysis, and ChIP assays. Results WT mice showed circadian rhythms in clock genes as well as mechanosensor, Cx26 and VNUT. Their expression was low during the sleep phase. The results of ChIP assays showed Clock protein binding to the promotor regions and the transcriptional regulation of mechanosensor, Cx26 and VNUT. In contrast, all of these circadian expressions were disrupted in ClockΔ19/Δ19 mice. The gene expression of mechanosensor, Cx26 and VNUT was maintained at a higher level in spite of the sleep phase. Conclusions Mechanosensor, Cx26 and VNUT expressed with circadian rhythm in the mouse bladder mucosa. The disruption of circadian rhythms in these genes, induced by the abnormalities in clock genes, may be factors contributing to NOC because of hypersensitivity to bladder wall extension.

The Clock mutant mouse is a novel experimental model for nocturia and nocturnal polyuria.

The pathophysiologies of nocturia (NOC) and nocturnal polyuria (NP) are multifactorial and their etiologies remain unclear in a large number of patients. Clock genes exist in most cells and organs, and the products of Clock regulate circadian rhythms as representative clock genes. Clock genes regulate lower urinary tract function, and a newly suggested concept is that abnormalities in clock genes cause lower urinary tract symptoms. In the present study, we investigated the voiding behavior of Clock mutant (ClockΔ19/Δ19) mice in order to determine the effects of clock genes on NOC/NP.

Multifocal metastases of recurrent renal cell carcinoma successfully treated with a combination of low dose interleukin‐2, α‐interferon and radiotherapy

Abstract  A 59‐year‐old man presented with a 2‐month history of left flank pain and a possibility of gross hematuria. Left renal cell carcinoma stage II was diagnosed and radical left nephrectomy was performed. Twenty‐two months postoperatively, lung metastases were demonstrated and 6 × 106 units of α‐interferon (IFN‐α) were administered for 9 months, only to keep the sizes of the metastases unchanged. Thirty‐four months after the operation, liver metastases and bone metastasis in the left sacroiliac joint were revealed. The combination cytokine therapy was performed with 1.4 × 106 U of interleukin‐2 (IL‐2) and 3 × 106 U of IFN‐α for 16 weeks, and the left sacroiliac joint metastasis was treated with radiation therapy of 4 Gy per day for 7 days. Six months after the 16 weeks of immunotherapy, computed tomography and bone scintigraphy revealed that the metastases of the lung, liver and bone substantially disappeared and this complete response is still kept after 16 months.

The oscillation of intracellular Ca 2+ influx associated with the circadian expression of Piezo1 and TRPV4 in the bladder urothelium

We previously showed that bladder functions are controlled by clock genes with circadian rhythm. The sensation of bladder fullness (SBF) is sensed by mechano-sensor such as Piezo1 and TRPV4 in the mouse bladder urothelium. However, functional circadian rhythms of such mechano-sensors remain unknown. To investigate functional circadian changes of these mechano-sensors, we measured circadian changes in stretch-evoked intracellular Ca2+ influx ([Ca2+]i) using mouse primary cultured urothelial cells (MPCUCs). Using Ca2+ imaging, stretch-evoked [Ca2+]i was quantified every 4 h in MPCUCs derived from wild-type (WT) and ClockΔ19/Δ19 mice, which showed a nocturia phenotype. Furthermore, a Piezo1 inhibitor GsMTx4 and a TRPV4 inhibitor Ruthenium Red were applied and stretch-evoked [Ca2+]i in MPCUCs was measured to investigate their contribution to SBF. Stretch-evoked [Ca2+]i showed a circadian rhythm in the WT mice. In contrast, ClockΔ19/Δ19 mice showed disrupted circadian rhythm. The administration of both GsMTx4 and Ruthenium Red eliminated the circadian rhythm of stretch-evoked [Ca2+]i in WT mice. We conclude that SBF may have a circadian rhythm, which is created by functional circadian changes of Piezo1 and TRPV4 being controlled by clock genes to be active during wakefulness and inactive during sleep. Abnormalities of clock genes disrupt SBF, and induce nocturia.

The Circadian expression of Piezo1, TRPV4, Connexin26, and VNUT, associated with the expression levels of the clock genes in mouse primary cultured urothelial cells

To investigate circadian gene expressions in the mouse bladder urothelium to establish an experimental model and study the functions of the circadian rhythm.