AMPK Suppresses Connexin43 Expression in the Bladder and Ameliorates Voiding Dysfunction in Cyclophosphamide-induced Mouse Cystitis.

Xiling Zhang,Jian Yao,Takahiko Mitsui,Kun Gao,Manabu Kamiyama,Tatsuya Ihara,Norifumi Sawada,Masayuki Takeda,Jianglin Fan,Yuan Chi

doi:10.1038/srep19708

Xiling Zhang, Jian Yao + Show 8 more

Open Access

https://doi.org/10.1038/srep19708

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Abstract

Bladder voiding dysfunction is closely related to local oxidation, inflammation, and enhanced channel activities. Given that the AMP-activated protein kinase (AMPK) has anti-oxidative, anti-inflammatory and channel-inhibiting properties, we examined whether and how AMPK affected bladder activity. AMPK activation in rat bladder smooth muscle cells (BSMCs) using three different AMPK agonists resulted in a decrease in connexin43 (Cx43) expression and function, which was associated with reduced CREB phosphorylation, Cx43 promoter activity and mRNA expression, but not Cx43 degradation. Downregulation of CREB with siRNA increased Cx43 expression. A functional analysis revealed that AMPK weakened BSMC contraction and bladder capacity. AMPK also counteracted the IL-1β- and TNFα-induced increase in Cx43 in BSMCs. In vivo administration of the AMPK agonist AICAR attenuated cyclophosphamide-initiated bladder oxidation, inflammation, Cx43 expression and voiding dysfunction. Further analysis comparing the responses of the wild-type (Cx43+/+) and heterozygous (Cx43+/−) Cx43 mice to cyclophosphamide revealed that the Cx43+/− mice retained a relatively normal micturition pattern compared to the Cx43+/+ mice. Taken together, our results indicate that AMPK inhibits Cx43 in BSMCs and improves bladder activity under pathological conditions. We propose that strategies that target AMPK can be developed as novel therapeutic approaches for treating bladder dysfunction.

Highlights

The cellular proteins were subjected to Western blot analysis for Cx43 and AMPK
The quantitative analysis indicated that incubating the Bladder smooth muscle cells (BSMCs) with 1 mM aminoimidazole-4-carboxamide-1-β -D-ribofuranoside (AICAR), 50 μ M flufenamic acid (FFA) or 5 mM metformin for 12 h led to a significant suppression of Cx43 protein expression (Fig. 1E)
Suppression of AMPK with a chemical inhibitor, Compound C, or a specific siRNA significantly enhanced Cx43 protein expression (Fig. 1F,G). These results suggest that AMPK activation inhibits Cx43 expression

Summary

Introduction

The cellular proteins were subjected to Western blot analysis for Cx43 and AMPK. A quantitative analysis of the effect of the AMPK siRNA on the levels of AMPK and Cx43 is shown in the lower panel of (Figure G). The pathological factors implicated in urinary disorders, such as oxidative stress, inflammatory mediators and growth factors, have been documented to be able to upregulate Cx43 expression[15,20,21,22,23,24,25] Because of these reasons, targeting Cx43 has been proposed as a potential approach to treat bladder hyperactivity. AMPK counteracts the biological actions of several inflammatory mediators and growth factors, such as interleukins, tumor necrosis factor, PDGF, etc., including those implicated in upregulation of Cx43 expression in the bladder and overactivity of bladder[21,22,28,30]. Our study indicates that treatments targeting AMPK can be developed as new therapeutic strategies to alleviate bladder overactivity

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